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Supplementary Figures 1-5 from Utilization of Pathway Signatures to Reveal Distinct Types of B Lymphoma in the Eμ-myc Model and Human Diffuse Large B-Cell Lymphoma

Supplementary Tables 6-9 from Utilization of Pathway Signatures to Reveal Distinct Types of B Lymphoma in the Eμ-myc Model and Human Diffuse Large B-Cell Lymphoma

Supplementary Tables 1-5 from Utilization of Pathway Signatures to Reveal Distinct Types of B Lymphoma in the Eμ-myc Model and Human Diffuse Large B-Cell Lymphoma

The authors declare that there are no disclosures or conflicts of interest regarding the publication of this manuscript. We did not receive any financial support and have no financial interests which could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.

Objectives Lineage switch occurs in rare leukemias, and the mechanism is unclear. We report two cases of B-lymphoblastic leukemia (B-ALL) relapsed as acute myeloid leukemia (AML). Methods Retrospective review of clinical and laboratory data. Results Complex cytogenetic abnormalities were detected in B-ALL for both cases with subclone heterogeneity. Postchemotherapy marrow biopsies showed trilineage hematopoiesis without detectable B-ALL. Cytogenetics in both showed stemline abnormalities. The cases were considered "occult" myelodysplastic syndrome (MDS) preceding B-ALL. The patients relapsed 6.5 and 9 months following induction, respectively. Case 1 relapsed as AML-M5 initially, was treated as such, and then relapsed again as B-ALL. Case 2 relapsed as AML-M6. Cytogenetics demonstrated persistent abnormalities. Both patients died soon after relapse. Conclusions Lineage switch between B-ALL and AML could be intermediated by occult MDS. A pluripotent progenitor likely undergoes neoplastic transformation, resulting in a genomically unstable clone. This leads to a repertoire of heterogeneous subclones that may be selected by chemotherapy. Lineage switch heralds a dismal clinical outcome.

As therapeutic options for various lymphomas have rapidly expanded in the last decade, accurate classification and prognostic stratification of common and uncommon lymphoma types are of utmost importance. The most recent WHO classification continues to emphasize morphology, immunophenotype, molecular genetic abnormalities, and clinical behavior as the necessary pillars for correct diagnosis. Surgical excisional biopsy (SEB) is both historically and currently the gold standard technique for morphological examination, but it requires more time and preparation, often with more extensive anesthesia with higher potential for medical complications. Because of this, elderly and/or frail patients may not be able to tolerate the stress of the procedure, particularly when abdominal, retroperitoneal, and thoracic masses are to be examined. Indeed, excisional biopsy may not be technically feasible in certain locations (e.g., retroperitoneal midline). Patients may want to avoid the discomfort and inconvenience associated with excisional biopsy and may prefer an alternative, even when it is not medically necessary. In response to these issues, over the last 25 years, there has been increasing utilization of fine needle aspiration (FNA) cytology and core needle biopsy (CNB) techniques for diagnostic screening of enlarged lymph nodes or other mass lesions suspected to harbor lymphomas, metastatic neoplasms, and reactive lymphadenopathies. We provide guidelines for appropriate handling of the limited specimens obtained by these “small biopsies” and suggest how to optimize the use of ancillary technics such as flow cytometry, immunohistochemistry (IHC), and molecular analysis, firstly, to differentiate reactive lesions from lymphomas and, secondly, to provide accurate classification and prognostic information. The ability to render a definite diagnosis according to the WHO classification on these “small biopsies” varies for different lymphoma types, and we discuss the limitations of these specimens for common lymphomas. In the second half of the chapter, the correct handling of small biopsies performed in previously diagnosed and treated patients is discussed. We emphasize the evaluation of these repeat biopsies for disease recurrence, disease progression, emergence of secondary pathological processes due to prior treatment, and presence of therapeutic targets for the second- and third-line treatments. The clinically crucial distinctions among lymphomas with overlapping features are mentioned, and guidance for issuing descriptive reports and for requesting additional material is provided.

A bone marrow biopsy sometimes demonstrates two hematolymphoid neoplasms. In a majority of these cases, a second neoplasm is discovered unexpectedly when examining the marrow for a known or suspected hematolymphoid neoplasm. There are several possible combinations of neoplastic components, but in practice, many cases show a myeloid neoplasm along with a nonmyeloid neoplasm such as plasma cell neoplasm (PCN), chronic lymphocytic leukemia (CLL), other small mature B-cell leukemia/lymphoma, T-cell large granular lymphocytic leukemia (T-LGL), or other T-cell lymphomas. The myeloid neoplasm, usually myelodysplastic syndrome, may occur as a consequence of therapy given for the lymphoid neoplasm, and such cases are covered in Chapter 25. In addition, two different nonmyeloid neoplasms can be seen together, including PCN with CLL, PCN with lymphoplasmacytic lymphoma, CLL with T-LGL, CLL with B-lymphoblastic leukemia, etc. Diagnosis of these concomitant hematolymphoid neoplasms is challenging in that one neoplastic component can potentially mask the other in the bone marrow examination. Here, we highlight the diagnostic approach to correctly identify more than one neoplastic components by using ancillary test on the marrow as well as other laboratory studies, discuss potential etiologies of the concurrency, and emphasize the clinical significance of the phenomenon for treatment and prognosis. Bone marrow involvement by solid tumors, with or without concurrent hematolymphoid neoplasm, is discussed in Chapter 28.

Key concepts in immunology intersect the practice of hematopathology because the biology and classification of lymphoid neoplasms are best understood on this basis and accurate diagnosis of most hematolymphoid neoplasms very often relies on immune-based techniques. This chapter summarizes the fundamental divisions of the immune system at the cellular and functional level and relates these divisions to the immune responses in health and disease. The normal development of B-cells and T-cells is mapped to the anatomical and histological distribution of these cells at various stages of maturation and through immune responses. The pros and cons of the two principal techniques of determining the immunophenotype of normal and abnormal cells—multiparameter flow cytometry and immunohistochemistry—are compared. The immunophenotype of normal lymphoid cell subsets, developing myeloid cells, and major types of lymphomas and acute leukemias are presented. Finally, the rapidly expanding repertoire of antibody based therapies for hematolymphoid maligngncies is highlighted with this background of key immunologic concepts.

Bone marrow examinations are done for a variety of benign and malignant conditions and are often triggered by abnormalities in the complete blood count and peripheral blood smear. In this chapter, a diagnostic approach to evaluating bone marrows in both adults and children will be reviewed. Optimal use of different aspects of the examination will be discussed as well as the use of ancillary testing. Finally, the organization of bone marrow reports and determination of when one can render a definitive versus a descriptive diagnosis will be reviewed.

Myeloid neoplasms occasionally occur in patients with sickle cell disease, and the underlying connection between the two diseases is unclear. Herein, we retrospectively analyzed four cases of sickle cell disease patients who developed myeloid neoplasm. Age at time of diagnosis ranged from 27 to 59 years with a median of 35.5 years. Two patients were treated with hydroxyurea and the other two with supportive care alone, with one out of the four patients receiving additional treatment with hematopoietic stem cell transplant. Three patients presented with leukocytosis, and the remaining patient presented with pancytopenia. Two patients were diagnosed with myelodysplastic syndrome/myeloproliferative neoplasm, one with myelodysplastic syndrome, and the other with acute myeloid leukemia. All four cases demonstrated certain degrees of myelodysplasia and complex cytogenetic abnormalities with - 7/7q- and/or - 5/5q- or with 11q23 (KMT2A) rearrangement. This cytogenetic profile resembles that seen in therapy-related myeloid neoplasm, suggesting that myeloid neoplasm in the setting of sickle cell disease may represent a subcategory of the disease distinct from de novo myeloid neoplasm in general. Extensive literature review further demonstrates this similarity in cytogenetic profile, as well as in other associated pathologic features. Potential etiology includes therapy for sickle cell disease, disease-related immunomodulation, or disease-related chronic inflammation. We hypothesize that constant hematopoietic hyperplasia, stimulated by a hemolysis-induced cytokine storm, may increase the chance of somatic mutations/cytogenetic aberrations, resulting in transformation of myeloid precursors. This group of myeloid neoplasms seems to herald a dismal clinical outcome, with median survival

Background Flow cytometric immunophenotyping (FCI) is recognized as a rapid, sensitive, and accurate method for diagnosis of B-cell lymphomas. We observed that FCI failed to identify the clonal B-cell population in several cases of large B-cell lymphoma (DLBCL) when tissue samples were prepared by a commercially available mechanical tissue disaggregation method. We tested a manual tissue disaggregation method and compared it with the mechanical method. Methods FCI findings from 51 cases of DLBCL processed with the mechanical tissue disaggregation method, 27 cases processed using the manual method, and 15 cases processed using a combination of both methods were compared. The histological and immunohistochemical findings in each case were reviewed. Results FCI detected a clonal B-cell population in 88.9% of cases processed by the manual tissue disaggregation method, 66.7% of cases processed by a combination of the manual and mechanical disaggregation methods, and in 62.7% of cases processed solely by the mechanical tissue disaggregation method (P

Background Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. Methods A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. Results/Conclusion Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 106 and 5 × 106 bone marrow cells to be measured, respectively. © 2015 International Clinical Cytometry Society

Objectives To compare the features of the blast phase of chronic myelogenous leukemia (CML) in patients treated with tyrosine kinase inhibitors (TKIs) with those in the pre-TKI era. Methods Sixty-seven patients with blast phase CML were identified in the Duke Pathology database from 1991 to 2011. The morphology and immunophenotype of blasts were evaluated, along with cytogenetic studies and associated findings in the peripheral blood and bone marrow. Results In the TKI era, the blasts were more frequently of a type other than the usual myeloid or lymphoid types when compared with the pre-TKI era. Blast phase in TKI-treated patients was associated with a higher peripheral WBC count and a lower blast percentage in the bone marrow. Of the 23 patients with cytogenetic studies during blast phase, additional cytogenetic changes more frequently occurred in patients with an unusual blast type, and some patients showed these changes months before the onset of blast phase. Conclusions Blast phase CML in TKI- and non-TKI-treated patients differs in the morphology and immunophenotype of blasts, cytogenetic findings, and associated findings in the peripheral blood and bone marrow.

Context.—Plasma cell myeloma and chronic lymphocytic leukemia are both common hematologic malignancies, sharing many epidemiologic features. Concomitant detection of the 2 conditions poses special diagnostic challenges for the pathologist. Objective.—To describe the pathologic findings in cases of concomitant bone marrow involvement by myeloma and CD5+ monoclonal B cells and to outline the differential diagnostic possibilities, suggest a workup for correct diagnosis, and examine clinical outcome. Design.—Fifteen cases that met the diagnostic criteria were identified from pathology databases at 4 participating institutions. Morphologic findings were reviewed, additional immunohistochemical stains performed, and flow cytometric, cytogenetic, and relevant laboratory and clinical information was summarized. Previously published cases were searched from electronic databases and cross-references. Results.—Most patients (13 of 15) were older males. Often (11 of 15) they presented clinically with myeloma, yet had both monotypic plasma cells and B cells in the diagnostic marrow. In 4 patients, myeloma developed 24 months or later after chronic lymphocytic leukemia. In 7 patients, myeloma and CD5+ B cells showed identical immunoglobulin light-chain restriction. Primary differential diagnoses include lymphoplasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia with plasmacytoid differentiation. CD56 and/or cyclin D1 expression by plasma cells was helpful for correct diagnosis. Most patients in our cohort and published reports were treated for plasma cell myeloma. Conclusions.—Concomitant detection of myeloma and chronic lymphocytic leukemia in the bone marrow is a rare event, which must be carefully differentiated from lymphomas with lymphoplasmacytic differentiation for correct treatment.

The decision to re-induce patients with acute myeloid leukemia (AML) based on results of the day 14 bone marrow (BM) biopsy is variable and lacks evidence based data. The aim of our review was to evaluate the accuracy of a day 14 BM biopsy in determining the need for re-induction chemotherapy.Seventy-four patients with newly diagnosed de novo AML treated with induction chemotherapy were retrospectively reviewed for the purpose of evaluating treatment decisions and outcomes based on their day 14 BM biopsy. Response to therapy in this analysis was based on morphology alone.Of the 74 patients undergoing standard induction, 45 patients (61%) had no evidence of leukemia on their day 14 BM biopsy. Eighteen patients (24%) had definitive residual disease (RD), and 11 patient's (15%) were classified as indeterminate response (IR). Fifteen patients with RD and one with IR underwent re-induction chemotherapy. However, thirteen patients (3 RD and 10 IR) were observed until count recovery without any re-induction therapy. Eleven of these 13 patients who were observed eventually attained a morphologic complete remission (CR), including two patients with RD.A day 14 BM biopsy may have suboptimal sensitivity for the detection of residual leukemia. Some patients with an IR on day 14 may not require re-induction chemotherapy, but instead, may benefit from careful observation until count recovery to avoid the mortality and morbidity associated with re-induction chemotherapy.

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Aims: To determine the roles of the presence of malignancy, tumour proliferation fraction, vascular compromise and therapeutic and diagnostic manipulations in lymph node infarction (LNI). Methods and results: Thirty-five cases of LNI were identified over a 20-year period. Of the 35 patients, 31 (89%) had an underlying malignancy: 27 of the 31 (87%) were haematologic malignancies, the rest being metastatic carcinoma (two), melanoma, and seminoma. Of the four patients without evidence of malignancy, two were diagnosed with viral infection, one had LNI adjacent to a thrombosed pancreas graft, and one was lost to follow-up. Ki67 immunostaining in viable tumour demonstrated a range (5–60%) of proliferation fractions. A history of fine needle aspiration alone was present in seven of the 35 patients (20%), a history of chemotherapy alone in 11 (31%), and a history of both in two (5.7%). Factor VIII immunostaining highlighted thrombosed and recanalized vessels next to the infarction. Conclusions: Infarction of lymph nodes is associated with previous, concurrent or subsequent diagnosis of malignancy in the vast majority of cases. Chemotherapy or previous fine needle aspiration can precipitate infarction in some cases, but infarction may occur without such intervention, possibly because of an underlying subacute or chronic vascular compromise produced by vascular thrombosis.

Objective Older adults with acute myeloid leukemia (AML) tend to have worse complete remission (CR) rates and overall survival compared to their younger counterparts. At least one reason for this is increased expression of the multidrug resistance gene (MDR1). Dose dense, high intensity chemotherapy may overcome the MDR1 effect, possibly when combined with anti-CD33 monoclonal antibody gemtuzumab ozogamicin (GO,Mylotarg™), which has been studied in older adults with relapsed AML. This phase I study was aimed at establishing safety by defining a maximum tolerated dose (MTD) by treating older AML patients with two cycles of dose-dense therapy with high dose cytarabine (HiDAC) combined with targeted therapy using GO. Materials and methods Nine patients ≥60years with newly diagnosed, untreated CD33+ AML with adequate renal and hepatic function, and ECOG PS 0-2 were eligible. HiDAC was administered at two dose levels: 3000mg/m 2 every 12h for 6 doses (cohort 1), or 9 doses (cohort 2). GO was administered at 6mg/m 2 on days 1 and 8. Results The MTD was HiDAC 3000mg/m 2 for six doses along with GO 6mg/m 2 . All patients had grades 3–4 pancytopenia, and two patients developed reversible grade 2 neurotoxicity. There were no cases of veno-occlusive disease. Seven of nine patients had a complete response (CR or CRp). Conclusions There was no difference in relapse-free survival in our patients when compared to historical data. However, despite high toxicity, two of nine patients treated in this dose-dense fashion remained in CR for >60months.

We present a case report of an 80-year-old woman with volume overload thought initially to be secondary to heart failure, but determined to be amiodarone-induced acute and chronic liver injury leading to submassive necrosis and bridging fibrosis consistent with early cirrhosis. Her histopathology was uniquely absent of steatosis and phospholipidosis, which are commonly seen in AIC.

We report 10 cases of donor cell leukemia (DCL). All cases except the case of chronic lymphocytic leukemia had anemia, neutropenia, and/or thrombocytopenia when DCL was diagnosed. Eight cases with sex-mismatched hematopoietic stem cell transplant (HCT) showed donor gonosomal complements, suggesting DCL. Clonal cytogenetic abnormalities were detected in 8 cases: 6 were monosomy 7/del(7q). In all 10 cases, engraftment studies confirmed donor cell origin. Retrospective fluorescence in situ hybridization in archived donor cells in 4 cases showed a low level of abnormalities in 2. Of 7 patients with clinical follow-up of 5 months or more, 1 (with acute myeloid leukemia) died of disease; 6 are alive, including 1 with myelodysplastic syndrome with spontaneous remission. Similar to reported cases, we found disproportional sex-mismatched HCTs, suggesting probable underdetection of DCL in sex-matched HCTs. The latency between HCT and DCL ranged from 1 to 193 months (median, 24 months), in keeping with the literature. Analyzing our cases, pooled with reported cases, with survival models showed much shorter latency for malignancy as primary disease, for T-cell large granular lymphocyte leukemia as type of DCL, and for umbilical cord blood as stem cell source.

Summary Histone H4 acetylation was examined by immunohistochemistry in patients with acute lymphocytic leukaemia (ALL) in first relapse. Univariate and multivariate models identified correlates of complete remission (CR) and overall survival (OS). No variables were associated with achievement of CR. In multivariate analysis, weak histone H4 acetylation [Hazard Ratio (HR) 2·20, 95% confidence interval (CI) 0·93–5·23, P = 0·07], shorter interval from diagnosis to relapse ( 24 months) (HR 1·82, 95% CI 1·20–2·75, P = 0·005), and central nervous system involvement (HR 3·43, 95% CI 1·31–8·99, P = 0·01) were independent poor prognostic factors for OS. These data provide a rationale for the use of histone deacetylase inhibitors in the treatment of relapsed ALL.

The molecular basis of the progression of chronic myeloid leukaemia from the chronic stage to the acute phase is poorly understood. Now, work in mouse models of chronic myeloid leukaemia shows that this progression is controlled by the cell fate regulator Musashi2, which in turn regulates Numb, Notch and p53 to block cellular differentiation. Musashi2 expression can be increased by aberrant transcription factors found in leukaemia and is observed during cancer progression in human patients with leukaemia, where it is associated with poorer prognosis. This raises the possibility that modulating Musashi–Numb associated signalling may serve as a new approach to therapies against this disease. Chronic myelogenous leukaemia (CML) can progress from a chronic to an acute phase. These authors show in mouse models that leukaemia progression is controlled by the cell-fate regulator Musashi2, which in turn regulates Numb, Notch and p53 to block cellular differentiation. Musashi2 expression can be increased by aberrant transcription factors found in leukaemia, is observed during cancer progression in human CML patients and is associated with poorer prognosis. Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase1, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML2,3 to show that disease progression is regulated by the Musashi–Numb signalling axis4,5. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML6,7, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Initial exposure of monocytes/macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Molecular mechanisms responsible for endotoxin tolerance are not well defined. We and others have shown that IL-1R–associated kinase (IRAK)-M and SHIP-1 proteins, negative regulators of TLR4 signaling, increase in tolerized cells. TGF-β1, an anti-inflammatory cytokine, is upregulated following LPS stimulation, mediating its effect through SMAD family proteins. Using a monocytic cell line, THP1, we show that LPS activates endogenous SMAD4, inducing its migration into the nucleus and increasing its expression. Secondary challenge with high dose LPS following initial low-dose LPS exposure does not increase IRAK-M or SHIP1 protein expression in small hairpin (sh)SMAD4 THP-1 cells compared with control shLUC THP1 cells. TNF-α concentrations in culture supernatants after second LPS challenge are higher in shSMAD4 THP-1 cells than shLUC THP1 cells, indicating failure to induce maximal tolerance in absence of SMAD4 signaling. Identical results are seen in primary murine macrophages and mouse embryonic fibroblasts, demonstrating the biological significance of our findings. TGF-β1 treatment does not increase IRAK-M or SHIP1 protein expression in shSMAD4 THP-1 cells, whereas it does so in shLUC THP1 cells, indicating that TGF-β1 regulates IRAK-M and SHIP1 expression through a SMAD4-dependent pathway. Knockdown of endogenous SHIP1 by shSHIP1 RNA decreases native and inducible IRAK-M protein expression and prevents development of endotoxin tolerance in THP1 cells. We conclude that in THP-1 cells and primary murine cells, SMAD4 signaling is required for maximal induction of endotoxin tolerance via modulation of SHIP1 and IRAK-M.

We compared the expression of CD123, the alpha chain of the interleukin-3 receptor, on normal B-cell precursors in bone marrow ("hematogones") from 75 specimens and on leukemic blasts in 45 newly diagnosed B-acute lymphoblastic leukemias (B-ALL) cases. We found that the less mature hematogones (dim CD45+) that express CD34 lack CD123 expression, whereas the more mature hematogones (moderate CD45+) lack CD34 but always express CD123. In contrast with this discordant pattern of CD34 and CD123 expression in hematogones, blasts in 41 (91%) of 45 cases of B-ALL showed concordant expression of the 2 antigens: 80% (36 of 45) cases expressed both antigens, whereas 11% (5 of 45) expressed neither. We found that these distinct patterns of CD34/CD123 expression on hematogones (discordant) and B-ALL blasts (concordant) remain stable after chemotherapy and are useful in differentiating small populations of residual blasts from hematogones that may be simultaneously present.

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

The IL-1 receptor-associated kinase 1 (IRAK-1) and IRAK-M are key signaling molecules in cellular responses to endotoxin initiated through the Toll-like receptors (TLRs). The aim of this study was to evaluate the effect of age on the modulation of TLRs and IRAK-1 and IRAK-M in peripheral blood mononuclear cells (PBMCs) exposed in vitro to endotoxin under conditions that could induce endotoxin tolerance. Peripheral blood mononuclear cells obtained from young (4- to 6-month-old) and aged (24- to 26-month-old) Brown Norway rats were treated with high-dose LPS, with or without priming with low-dose LPS. In comparison with younger rats, the intensity of TLR-4 expression was persistently high in monocytes from aged rats after stimulation with LPS and was not decreased by priming with low-dose LPS (P0.05). Messenger RNA (mRNA) for TLR-4 in PBMCs from aged rats did not show any decrease after priming with low-dose LPS as seen in PBMCs from young rats at 24 h (P = 0.01) after restimulation. In PBMCs from young rats, but not aged rats, preconditioning with low-dose LPS and subsequent stimulation with high-dose LPS resulted in markedly decreased IRAK-1 protein (P = 0.02) and decreased mRNA for IRAK-1 (P0.05). In contrast, PBMCs from aged rats treated in this manner continued to express measurable levels of IRAK-1 protein. Preconditioning with low-dose LPS caused an increase in both IRAK-M protein and mRNA (P = 0.05) after stimulation with high-dose LPS only in cells from young rats. These phenotypic characteristics of PBMCs from aged rats can interfere with their ability to develop tolerance to endotoxin.

The Eμ-myc transgenic mouse has provided a valuable model for the study of B-cell lymphoma. Making use of gene expression analysis and, in particular, expression signatures of cell signaling pathway activation, we now show that several forms of B lymphoma can be identified in the Eμ-myc mice associated with time of tumor onset. Furthermore, one form of Eμ-myc tumor with pre-B character is shown to resemble human Burkitt lymphoma, whereas others exhibit more differentiated B-cell characteristics and show similarity with human diffuse large B-cell lymphoma in the pattern of gene expression, as well as oncogenic pathway activation. Importantly, we show that signatures of oncogenic pathway activity provide further dissection of the spectrum of diffuse large B-cell lymphoma, identifying a subset of patients who have very poor prognosis and could benefit from more aggressive or novel therapeutic strategies. Taken together, these studies provide insight into the complexity of the oncogenic process and a novel strategy for dissecting the heterogeneity of B lymphoma. [Cancer Res 2008;68(20):8525–34]

Context.—Marginal zone lymphomas (MZLs) are the most common lymphomas encountered in the orbit and ocular adnexa. The accurate categorization of these lymphomas is critical to avoid undertreatment or overtreatment. Objective.—To identify features of orbital MZLs that distinguish them from other lymphomas and reactive lymphoid infiltrates and support the categorization of orbital MZL as mucosa-associated lymphoid tissue (MALT)–type MZLs. Design.—Biopsies from 149 patients with lymphoid lesions of ocular adnexa were examined. Additional immunohistochemical stains and fluorescence in situ hybridization study for the MALT1 locus were performed in selected cases, and patient charts were reviewed. Results.—A total of 115 lymphomas and 34 reactive infiltrates were identified, of which B-cell lymphomas constituted 92% and MZLs constituted 54% of all lymphomas. Certain clinical features (young age, race, bilaterality) favored a reactive infiltrate, but none were diagnostic. Histologic features, such as infiltrative lesions, reactive B-cell follicles, and lymphoepithelial lesions, overlapped between reactive infiltrates and conjunctival MZL. In contrast to conjuctival MZL, orbital MZL infrequently showed reactive follicles, rarely showed epithelial tissue, and did not show lymphoepithelial lesions. Cytogenetic abnormality involving the MALT1 locus was demonstrated in only 15% of ocular adnexal MZLs. Conclusion.—Many MZLs of orbital soft tissue lack key features associated with MALT-type MZL, and the designation MALT lymphoma should be avoided in their diagnosis.

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.

A 4-year-old African American male was referred to the Pediatric Neurosurgery Service for evaluation of new onset seizures and worsening mental status. An MRI of the brain revealed a pineal region mass with diffuse leptomeningeal enhancement and compression of the basilar cisterns. A biopsy of the brain revealed histologic and immunophenotypic findings characteristic of ALK-1+ anaplastic large cell lymphoma (ALCL). ALCL rarely occurs in the central nervous system and poses a significant diagnostic challenge often leading to a delay in the initiation of appropriate treatment. We describe a case of a rapidly deteriorating clinical course in a child with central nervous system ALCL and review the current literature on ALCL occurring in the central nervous system.

Context.—Nonmyeloablative stem cell transplantation (NMSCT) is a mode of immunotherapy increasingly employed in treating hematologic, lymphoid, and solid tumors. Patients are monitored principally by molecular analysis of donor engraftment. Objective.—To determine the role of morphologic examination of bone marrow after NMSCT. Design.—Seventy-three patients undergoing NMSCT under the Campath 1H (humanized anti-CD52 antibody) protocol were studied. Pretransplant and sequential posttransplant bone marrow specimens were evaluated and the findings were correlated with corresponding engraftment data. Results.—Pretransplant bone marrow specimens from 43% of the patients were involved by disease, and these marrow specimens were significantly more cellular than those that were free of disease. Morphologically detectable disease was still present in day 14 posttransplant marrow specimens in more than one half of these patients, but there was no difference in engraftment in those with or without marrow disease. Early posttransplant marrow in nearly one half of the patients showed myeloid hyperplasia and atypical localization of immature myeloid precursors. Marrow cellularity for the first 2 months after NMSCT was significantly lower in those patients receiving stem cells mismatched at 1 to 3 loci as compared with those who received fully matched grafts (mean cellularity, 38.1% vs 54.1% at day 14). Marrow failure without recurrent disease at 3 to 6 months after transplant was detected by engraftment study in only approximately 15% of cases. Similarly, early recurrence of disease was detected first by morphologic examination in 4 of 13 cases before a decline in donor engraftment occurred. Conclusion.—Morphologic examination of bone marrow provides additional information that is complementary to donor engraftment analysis for optimal management after NMSCT.

Reported are 7 cases of posttransplant lymphoproliferative disorder (PTLD) arising in children who received umbilical cord blood transplantation (UCBT). There were 4 females and 3 males with a median age of 3 years (range, 1-16 years). All 7 patients received UCBT, including 1 patient who received multiple units and 1 transplanted under nonmyeloablative condition. The time interval from UCBT to PTLD averaged 4 months (range, 2 weeks to 9 months). Patients typically presented with high-stage disease with visceral organ involvement. Histology of the PTLDs showed monomorphic morphology in 5 cases and polymorphic morphology in the remaining 2 cases. Bone marrow biopsies were performed in 3 cases and were negative for PTLD. Epstein-Barr virus (EBV) was detected in the PTLD in all 7 patients by in situ hybridization. Evidence of past EBV infection was found in the recipients, but the EBV genome was not detected in the donor cord blood samples, suggesting that donor cord blood was not the source of EBV infection. The origin of the PTLD was investigated in 5 cases. PTLD was of host origin in 2 patients who failed engraftment and of donor origin in the remaining 3 patients who had complete engraftment. Four of 5 patients with monomorphic PTLD failed to demonstrate significant responses to rituximab and/or reduction of immunosuppression and died within 1 month after diagnosis. The remaining 2 patients with polymorphic PTLD showed complete response to therapy. One patient was alive 35 months after transplant, and the other patient died of infection 6 months after transplant. It is concluded that PTLD arising after UCBT in children occurs early after transplant and represents a serious EBV-related complication. PTLD may be of donor or recipient origin depending on engraftment status. Both monomorphic and polymorphic histology may be seen, and monomorphic histology appears to predict an unfavorable prognosis.

Posttransplant lymphoproliferative disorders (PTLDs) represent a spectrum ranging from Epstein-Barr virus (EBV)-driven polyclonal lymphoid proliferations to EBV+ or EBV- malignant lymphomas. Central nervous system (CNS) PTLDs have not been characterized fully. We reviewed the clinical, radiologic, and pathologic features of 12 primary CNS PTLDs to define them more precisely. Patients included 10 males and 2 females (median age, 43.4 years) who were recipients of kidney (n = 5), liver (n = 2), heart (n = 2), peripheral blood stem cells (n = 2), or bone marrow (n = 1). All received immunosuppressive therapy. CNS symptoms developed 3 to 131 months (mean, 31 months) after transplantation. By neuroimaging, most showed multiple (3 to 9) intra-axial, contrast-enhancing lesions. Histologic sections showed marked expansion of perivascular spaces by large, cytologically malignant lymphoid cells that were CD45+, CD20+, EBV+ and showed light chain restriction or immunoglobulin gene rearrangement. In distinction to PTLDs in other organ systems, CNS PTLDs were uniformly high-grade lymphomas that fulfilled the World Health Organization criteria for monomorphic PTLDs. Extremely short survival periods were noted for each CNS PTLD that followed peripheral blood stem cell transplantation. Survival of others with CNS PTLD varied; some lived more than 2 years.

We retrospectively reviewed 74 fine-needle aspiration (FNA) cases of presumptive non-Hodgkin lymphoma (NHL). All the cases had cytology and core-needle biopsy and 53 cases had concurrent flow cytometric analysis. FNA (cytology and flow cytometry) and core-needle biopsy were evaluated independently. FNA was diagnostic of diffuse large B-cell lymphoma (DLBL) in 25% (13/53) of cases and small B-cell NHL in 15% (8/53) of cases, whereas core-needle biopsy was diagnostic of DLBL in 37% (27/74) of cases and small B-cell NHL in 8% (6/74) of cases. Subclassification of small B-cell NHL was reached in 3/6 cases by core-needle biopsy. Insufficient cases were observed in both FNA (47%; 25/53) and core-needle biopsy (28%; 21/74) groups. With the combination of FNA and core-needle biopsy, diagnostic cases of DLBL increased to 43% (32/74) and insufficient samples were reduced to 16% (12/74). There was no clear advantage in the diagnosis and classification of small B-cell NHL by adding core-needle biopsy to FNA (14%; 10/74). We conclude that core-needle biopsy is a useful adjunct to FNA in the diagnosis of DLBL and shall be encouraged. In small B-cell NHL, core-needle biopsy does not add to the diagnostic ability of FNA. Cases insufficient for diagnosis may be seen in both core-needle biopsy and FNA. A combined approach reduces the number of insufficient cases and is recommended in routine FNA practice.

We report five cases of Burkitt lymphoma arising in organ transplant recipients. There were four men and one woman with a mean age of 35 years. All were solid organ recipients with three renal, one liver, and one double lung transplantation. The time interval between organ transplantation and lymphoma averaged 4.5 years. Patients typically presented with high-stage disease with generalized lymphadenopathy and bone marrow involvement. Histology showed classic Burkitt lymphoma or atypical variant/Burkitt-like morphology. C-MYC rearrangement, including three cases with immunoglobulin heavy chain and two cases with lambda light chain, and Epstein-Barr virus were detected in all the cases. Additional chromosomal abnormalities were present in two of three cases and p53 mutation was found in one of three cases. Aberrant genotype and phenotype were frequently encountered, including minor monoclonal or oligoclonal T-cell populations and undetectable surface immunoglobulin light chain expression. Four patients received antilymphoma regimens, with combination chemotherapy (three patients) and/or Rituximab (three patients), in addition to reduction of immunosuppression. All four patients achieved complete remission. We conclude that posttransplant Burkitt lymphoma represents a characteristic clinicopathologic entity and occurs later after transplantation. Genotypic and phenotypic aberrations are often present. Rituximab may be an effective alternative to conventional combination chemotherapy in the treatment of a posttransplant Burkitt lymphoma.

To compare the features of the blast phase of chronic myelogenous leukemia (CML) in patients treated with tyrosine kinase inhibitors (TKIs) with those in the pre-TKI era.Sixty-seven patients with blast phase CML were identified in the Duke Pathology database from 1991 to 2011. The morphology and immunophenotype of blasts were evaluated, along with cytogenetic studies and associated findings in the peripheral blood and bone marrow.In the TKI era, the blasts were more frequently of a type other than the usual myeloid or lymphoid types when compared with the pre-TKI era. Blast phase in TKI-treated patients was associated with a higher peripheral WBC count and a lower blast percentage in the bone marrow. Of the 23 patients with cytogenetic studies during blast phase, additional cytogenetic changes more frequently occurred in patients with an unusual blast type, and some patients showed these changes months before the onset of blast phase.Blast phase CML in TKI- and non-TKI-treated patients differs in the morphology and immunophenotype of blasts, cytogenetic findings, and associated findings in the peripheral blood and bone marrow.

Castleman disease is a rare lymphoproliferative disorder of unknown etiology that most commonly presents as a mediastinal nodal mass or, in the extranodal form of the disease, a mass located in the mediastinum or retroperitoneum. It is exceptionally uncommon for Castleman disease to present in the extremities. We report a rare case of extranodal Castleman disease presenting as a muscular forearm mass. We compare our case with the seven other reported cases in which Castleman disease presented as an isolated soft tissue mass in the extremities.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many morphologic and immunophenotypic features. In addition to histomorphologic examination, it is customary to use the absence of CD23 to differentiate MCL from CLL/SLL, based primarily on reported comparisons of immunohistochemical staining of tissue sections. These findings are widely extrapolated to flow cytometric analysis, although available data are contradictory and not sufficiently detailed. We compared expression of CD23 by flow cytometry in 22 cases of MCL and 25 cases of CLL/SLL. Lymphoma cells in 12 of 22 MCLs were negative for CD23, and 10 showed dim expression. In contrast, none of 25 CLL/SLLs were negative for CD23, 4 were dimly positive, and 21 were moderately or brightly positive. Thus, a significant proportion of MCL exhibited overlap of CD23 expression in the low-intensity range with CLL/SLL. Clinically, there was no correlation between the intensity of CD23 expression and clinical stage at diagnosis or survival. These findings emphasize that by flow cytometry, MCL can be differentiated reliably from CLL/SLL using CD23 if negative expression is observed. However, with dimly positive expression, interpretation should be cautious.

The objective of this study was to examine the effects of ursodeoxycholic (UDCA) and chenodeoxycholic acid (CDCA) on autoimmune disease in the NZBNZW F1 (B =W) mouse model of systemic lupus erythematosus (SLE). The development of murine lupus was assessed in female B/W mice given UDCA or CDCA. At 6 week intervals mice were examined for weight change, albuminuria, anti-DNA antibody and total IgG levels. Morbidity and mortality were assessed daily. UDCA-and CDCA-treated mice were examined at 24 weeks of age for serum cytokines, lymphocyte phenotype, and in vitro cytokine production after immunization with DNP-KLH. Liver and kidneys were examined histopathologically. The administration of UDCA and CDCA was tolerated without side effects. Weight gain in UDCA-or CDCA-treated and control mice was identical through 24 weeks of age. CDCA, but not UDCA, suppressed the development of renal disease. CDCA-treated B/W mice also had improved survival compared to UDCA-treated or control B/W mice. There were no significant effects of CDCA on anti-DNA antibodies, serum total IgG, or other immunologic parameters. CDCA-treated mice had lower serum IFN-g concentrations compared to control and UDCA-treated mice. The bile acid, CDCA, significantly inhibited the development of renal disease and modestly prolonged lifespan in the female B/W mouse model of SLE. Suppression of glomerulonephritis was associated with lower serum IFNg concentrations. Further investigation is needed to verify potential mechanisms of action, but these findings suggest that bile acids may alter the development or progression of autoimmunity.

Inflammation produces reactive oxygen intermediates (ROI) that cause vascular damage and activate T lymphocytes. Conversely, antioxidants not only protect tissue from oxidative damage but also suppress immune reactivity. The objective of this study was to examine immunomodulatory effects of the non-enzymatic antioxidants, N-acetylcysteine (NAC) and cysteamine (CYST), on autoimmune disease, glomerulonephritis, and mortality in the female B/W mouse model of human systemic lupus erythematosus (SLE). The development of murine lupus was assessed during the lifespan of female B/W mice given NAC or CYST. Morbidity and mortality were assessed daily. At 6 week intervals mice were examined for weight change, albuminuria, serum BUN, antibodies to DNA, and IgG immunoglobulin levels. Serum prolactin, estrogen and progesterone were measured at 18 weeks of age. In a parallel study, NAC-and CYST-treated and control B/W mice were examined at 24 weeks of age for interval renal histopathology, lymphocyte adhesion molecule expression, and antibody titers and in vitro cytokine production in response to immunization with DNP-KLH. CYST significantly suppressed development of albuminuria and azotemia at 36 and 42 weeks of age compared to control and NAC-treated mice. NAC significantly suppressed anti-DNA antibody levels at 24 weeks. In contrast CYST significantly increased anti-DNA antibody levels at 18 weeks of age (P < 0.001 CYST vs control and NAC-treated mice). Kidneys of CYST-treated mice also had accelerated inflammatory histologic changes despite their lower incidence of albuminuria and azotemia. Mean (± s.e.m.) survival of control mice was 33±2 weeks compared to 38±2 weeks in NAC-treated mice (P < 0.05 vs control), and 48±2 weeks in the CYST-treated group (P < 0.01 vs control mice). The antioxidants, NAC and CYST, significantly improved mortality in the female B/W mouse model of SLE. NAC suppressed autoantibody formation and modestly prolonged survival. CYST, despite its augmentation of anti-DNA levels and renal inflammatory changes, inhibited the development of renal insufficiency and markedly improved survival. These findings suggest that ROIs play a role in the pathogenesis of lupus nephritis and that antioxidants reduce the damage causing renal insufficiency. Antioxidants may be a beneficial adjunctive therapy in the treatment of human SLE.

Sex and sex hormones modulate immune development and responses. A primary target of their effects is the structure and cellularity of the thymus; therefore, we examined the effects of sex and sex steroids on thymocyte apoptosis. We demonstrate initially that male DBA mice have a significantly higher percentage of glucocorticoid-induced apoptotic thymocytes (46.1+/-3.8%) than their female counterparts (31.6+/-3.1%; P=0.012). We postulated that this gender difference was due to differential modulation of glucocorticoid-induced apoptosis by sex hormones such as estrogen, testosterone or progesterone. Both estrogen and testosterone increased in vitro thymocyte apoptosis. In contrast, progesterone not only inhibited spontaneous in vitro thymocyte apoptosis, but also prevented in vitro glucocorticoid-induced apoptosis. Progesterone administration also suppressed glucocorticoid-induced in vivo thymocyte apoptosis. These results suggest that anti-apoptotic effects of progesterone may influence T cell development and subsequent immune responses.

Background. In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival.The role of endothelial cell alteration in chronic rejection was examined in our model. Methods. Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. Results. Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. Conclusion. Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-gamma/TNF-alpha-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.

The most common identifiable causes of acute liver failure in pediatric patients are infection, drug toxicity, metabolic disease, and autoimmune processes. In many cases, the etiology of acute liver failure cannot be determined. Acute leukemia is an extremely rare cause of acute liver failure, and liver transplantation has traditionally been contraindicated in this setting. We report a case of acute liver failure in a previously healthy 15-yr-old male from pre-B-cell acute lymphoblastic leukemia. He underwent liver transplantation before the diagnosis was established, and has subsequently received chemotherapy for pre-B-cell acute lymphoblastic leukemia. He is currently alive 31 months post-transplantation. The published literature describing acute lymphoblastic leukemia as a cause of acute liver failure is reviewed.