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T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic neoplasm. Differentiation stage and immune-effector functions of the underlying tumor cell are insufficiently characterized. Constitutive activation of the T-cell leukemia 1A (TCL1A) oncogene distinguishes the (pre)leukemic cell from regular postthymic T cells. We assessed activation-response patterns of the T-PLL lymphocyte and interrogated the modulatory impact by TCL1A. Immunophenotypic and gene expression profiles revealed a unique spectrum of memory-type differentiation of T-PLL with predominant central-memory stages and frequent noncanonical patterns. Virtually all T-PLL expressed a T-cell receptor (TCR) and/or CD28-coreceptor without overrepresentation of specific TCR clonotypes. The highly activated leukemic cells also revealed losses of negative-regulatory TCR coreceptors (eg, CTLA4). TCR stimulation of T-PLL cells evoked higher-than-normal cell-cycle transition and profiles of cytokine release that resembled those of normal memory T cells. More activated phenotypes and higher TCL1A correlated with inferior clinical outcomes. TCL1A was linked to the marked resistance of T-PLL to activation- and FAS-induced cell death. Enforced TCL1A enhanced phospho-activation of TCR kinases, second-messenger generation, and JAK/STAT or NFAT transcriptional responses. This reduced the input thresholds for IL-2 secretion in a sensitizer-like fashion. Mice of TCL1A-initiated protracted T-PLL development resembled such features. When equipped with epitope-defined TCRs or chimeric antigen receptors, these Lckpr-hTCL1Atg T cells gained a leukemogenic growth advantage in scenarios of receptor stimulation. Overall, we propose a model of T-PLL pathogenesis in which TCL1A enhances TCR signals and drives the accumulation of death-resistant memory-type cells that use amplified low-level stimulatory input, and whose loss of negative coregulators additionally maintains their activated state. Treatment rationales are provided by combined interception in TCR and survival signaling., (© 2020 by The American Society of Hematology.)

The genetic background of follicular lymphomas (FLs) diagnosed in advanced clinical stages III/IV, and which are frequently characterized by t(14;18), has been substantially unraveled. Molecular features, as exemplified in the clinicogenetic risk model m7FLIPI, are important tools in risk stratification. In contrast, little information is available concerning localized-stage FL (clinical stages I/II), which accounts for ∼20% of newly diagnosed FL in which the detection rate of t(14;18) is only ∼50%. To investigate the genetic background of localized-stage FL, patient cohorts with advanced-stage FL or localized-stage FL, uniformly treated within phase 3 trials of the German Low-Grade Lymphoma Study Group, were comparatively analyzed. Targeted gene expression (GE) profiling of 184 genes using nCounter technology was performed in 110 localized-stage and 556 advanced-stage FL patients. By penalized Cox regression, a prognostic GE signature could not be identified in patients with advanced-stage FL, consistent with results from global tests and univariate regression. In contrast, it was possible to define robust GE signatures discriminating localized-stage and advanced-stage FL (area under the curve, 0.98) by penalized logistic regression. Of note, 3% of samples harboring an "advanced-stage signature" in the localized-stage cohort exhibited inferior failure-free survival (hazard ratio [HR], 7.1; P = .0003). Likewise, in the advanced-stage cohort, 7% of samples with a "localized-stage signature" had prolonged failure-free survival (HR, 2.3; P = .017) and overall survival (HR, 3.4; P = .072). These data support the concept of a biological difference between localized-stage and advanced-stage FL that might contribute to the superior outcome of localized FL., (© 2020 by The American Society of Hematology.)

To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper- N -glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach., (© 2018 by The American Society of Hematology.)

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP , TNFRSF14 / HVEM , and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D- mutated cases harbored multiple mutations in KMT2D , as compared with only 12% in LSTFL ( P = .019) and 0% in DTFL ( P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease., (© 2018 by The American Society of Hematology.)

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL., (© 2016 by The American Society of Hematology.)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) accounts for approximately 5% of all Hodgkin lymphoma cases. The aim of this study was to evaluate the prognostic implication of histopathologic NLPHL variants. Biopsies of 423 NLPHL patients treated within 9 prospective clinical trials performed by the German Hodgkin Study Group were classified as tumor cell-rich cases (n = 10), typical NLPHL (n = 308), or histopathologic variants (n = 105). Histopathologic variants were characterized by the presence of lymphoma cells outside the B-cell nodules or B-cell depletion of the microenvironment. Compared with typical NLPHL, histopathologic variants were associated with advanced disease (29.5% vs 14.6%, P = .0012) and a higher relapse rate (18.1% vs 6.5% at 5 years, P = .0009). Variant histology represented an independent prognostic factor (odds ratio = 2.955) in a multivariate model of progression/relapse. A prognostic score, including the risk factors variant histopathologic growth pattern, low serum albumin, and male gender, was derived from this model and allowed the definition of 3 distinct risk groups. NLPHL patients presenting with histopathologic variants have a poorer outcome compared with those showing typical histology. The newly developed prognostic score combining histologic and clinical features allows allocating NLPHL patients to defined risk groups.

MYC rearrangements occur in 5% to 10% of diffuse large B-cell lymphomas (DLBCL) and confer an increased risk to cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone (CHOP) and rituximab (R)-CHOP treated patients. We investigated the prognostic relevance of MYC-, BCL2- and BCL6-rearrangements and protein expression in a prospective randomized trial. Paraffin-embedded tumor samples from 442 de novo DLBCL treated within the RICOVER study of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry and fluorescence in situ hybridization (FISH) to detect protein expression and breaks of MYC, BCL2, and BCL6. Rearrangements of MYC, BCL2, and BCL6 were detected in 8.8%, 13.5%, and 28.7%, respectively. Protein overexpression of MYC (>40%) was encountered in 31.8% of tumors; 79.6% and 82.8% of tumors expressed BCL2 and BCL6, respectively. MYC translocations, MYChigh, BCL2high, and BCL6low protein expressions were associated with inferior survival. In multivariate Cox regression modeling, protein expression patterns of MYC, BCL2 and BCL6, and MYC rearrangements were predictive of outcome and provided prognostic information independent of the International Prognostic Index (IPI) for overall survival and event-free survival. A combined immunohistochemical or FISH/immunohistochemical score predicts outcome in DLBCL patients independent of the IPI and identifies a subset of 15% of patients with dismal prognosis in the high-risk IPI group following treatment with R-CHOP. Registered at http://www.cancer.gov/clinicaltrials: RICOVER trial of the DSHNHL is NCT 00052936.

The pathogenesis of classical Hodgkin lymphoma (cHL), the most common lymphoma in the young, is still enigmatic, largely because its Hodgkin and Reed-Sternberg (HRS) tumor cells are rare in the involved lymph node and therefore difficult to analyze. Here, by overcoming this technical challenge and performing, for the first time, a genome-wide transcriptional analysis of microdissected HRS cells compared with other B-cell lymphomas, cHL lines, and normal B-cell subsets, we show that they differ extensively from the usually studied cHL cell lines, that the lost B-cell identity of cHLs is not linked to the acquisition of a plasma cell-like gene expression program, and that Epstein-Barr virus infection of HRS cells has a minor transcriptional influence on the established cHL clone. Moreover, although cHL appears a distinct lymphoma entity overall, HRS cells of its histologic subtypes diverged in their similarity to other related lymphomas. Unexpectedly, we identified 2 molecular subgroups of cHL associated with differential strengths of the transcription factor activity of the NOTCH1, MYC, and IRF4 proto-oncogenes. Finally, HRS cells display deregulated expression of several genes potentially highly relevant to lymphoma pathogenesis, including silencing of the apoptosis-inducer BIK and of INPP5D, an inhibitor of the PI3K-driven oncogenic pathway.

The survival of diffuse large B-cell lymphoma patients varies considerably, reflecting the molecular diversity of tumors. In view of the controversy whether cytologic features, immunohistochemical markers or gene expression signatures may capture this molecular diversity, we investigated which features provide prognostic information in a prospective trial in the R-CHOP treatment era. Within the cohort of DLBCLs patients treated in the RICOVER-60 trial of the German High-Grade Lymphoma Study Group (DSHNHL), we tested the prognostic impact of IB morphology in 949 patients. The expression of immunohistochemical markers CD5, CD10, BCL2, BCL6, human leukocyte antigen (HLA)-DR, interferon regulatory factor-4/multiple myeloma-1 (IRF4/MUM1), and Ki-67 was assessed in 506 patients. Expression of the immunohistochemical markers tested was of modest, if any, prognostic relevance. Moreover, the Hans algorithm using the expression patterns of CD10, BCL6, and interferon regulatory factor-4/multiple myeloma-1 failed to show prognostic significance in the entire cohort as well as in patient subgroups. IB morphology, however, emerged as a robust, significantly adverse prognostic factor in multivariate analysis, and its diagnosis showed a good reproducibility among expert hematopathologists. We conclude, therefore, that IB morphology in DLBCL is likely to capture some of the adverse molecular alterations that are currently not detectable in a routine diagnostic setting, and that its recognition has significant prognostic power.

The transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T-cell transcription factor GATA3 is observed in B cell-derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidate the regulation and function of this factor in HL. We demonstrate binding of NFκB and Notch-1, 2 factors with deregulated activity in HL to GATA3 promoter elements. Interference with NFκB and Notch-1 activity led to decreased GATA3 expression, indicating a dependency of deregulated GATA3 expression on these transcription factors. Down-regulation of GATA3 in HL cell lines demonstrated its role in the regulation of IL-5, IL-13, STAT4, and other genes. A correlation between GATA3 and IL-13 expression was confirmed for HRS cells in HL tissues. Thus, GATA3 shapes the cytokine expression and signaling that is typical of HL. Conclusively, aberrant GATA3 expression in HRS cells is stimulated by the deregulated constitutive activity of NFκB and Notch-1, indicating a complex network of deregulated transcription factors in these cells. GATA3 activity significantly contributes to the typical cytokine secretion of and signaling in HRS cells, which presumably plays an essential role in HL pathogenesis.

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental "worst case scenario," we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or DeltaTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1-deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

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

Aberrant activities of JAK/STAT signaling pathways have been observed in several hematologic malignancies. Here, we show high expression of JAK2 in the tumor cells of lymphocyte-predominant Hodgkin lymphoma in 85% of cases and activation of JAK2 in 39% of cases. STAT6, which is a target of JAK2, was activated in 50% of the cases. SOCS1 controls JAK2 activity and degradation. Mutations in SOCS1 of either somatic or germ-line origin were observed in micromanipulated tumor cells of 50% of cases. Most mutations truncated SOCS1 or caused replacement of amino acids in functional important regions. Activating mutations in exon 12 of JAK2, which are frequent in myeloproliferative diseases, were not observed. In lymphocyte-predominant Hodgkin lymphoma SOCS1 function may thus be frequently impaired by mutations, and this may contribute to high JAK2 expression and activation of the JAK2/STAT6 pathway.

Transmembrane adaptor proteins (of which 7 have been identified so far) are involved in receptor signaling in immune cells. They have only a short extracellular region, with most of the molecule comprising a substantial intracytoplasmic region carrying multiple tyrosine residues that can be phosphorylated by Src- or Syk-family kinases. In this paper, we report an immunohistologic study of 6 of these molecules in normal and neoplastic human tissue sections and show that they are restricted to subpopulations of lymphoid cells, being present in either T cells (LAT, LIME, and TRIM), B cells (NTAL), or subsets of both cell types (PAG and SIT). Their expression in neoplastic lymphoid cells broadly reflects that of normal lymphoid tissue, including the positivity of plasma cells and myeloma/plasmacytoma for LIME, NTAL, PAG, and SIT. However, this study also revealed some reactions that may be of diagnostic/prognostic value. For example, lymphocytic lymphoma and mantle-cell lymphoma showed similar profiles but differed clearly from follicle-center lymphoma, whereas PAG tended to be selectively expressed in germinal center-derived subsets of diffuse large B-cell lymphoma. These molecules represent a potentially important addition to the panel of immunophenotypic markers detectable in routine biopsies that can be used in hematopathologic studies.

The pathogenesis of Hodgkin lymphoma (HL) is still largely unknown. Based on a search for footprints of pathogenetic mechanisms in global RNA expression data of Hodgkin/Reed-Sternberg (HRS) cell lines, we analyzed the expression and activation of 6 receptor tyrosine kinases (RTKs) in classic HL. Immunohistochemistry revealed that the RTKs platelet-derived growth factor receptor A (PDGFRA), DDR2, EPHB1, RON, TRKB, and TRKA were each expressed in HRS cells in 30% to 75% of patients. These RTKs were not expressed in normal B cells, the origin of HRS cells, or in most B-cell non-Hodgkin lymphoma (NHL). In the majority of patients at least one RTK was expressed, and in most patients several RTKs were coexpressed, most prominently in Hodgkin lymphoma of the nodular sclerosis subtype. Phosphotyrosine-specific antibodies revealed exemplarily the activation of PDGFRA and TRKA/B and an elevation of cellular phosphotyrosine content. Immunohistochemistry for RTK ligands indicated that DDR2 and TRKA are likely activated in a paracrine fashion, whereas PDGFRA and EPHB1 seem to be activated by autocrine loops. Activating mutations were not detected in cDNA encoding the RTKs in HRS cell lines. These findings show the unprecedented coexpression of multiple RTKs in a tumor and indicate that aberrant RTK signaling is an important factor in HL pathogenesis and that it may be a novel therapeutic target.

The neoplastic cells in classical Hodgkin disease (Reed-Sternberg cells) are of B-lymphoid origin, but they lack many markers of this cell lineage, for example, immunoglobulin, CD20, and B-cell-associated transcription factors. In contrast, the neoplastic cells ("L&H" cells) in lymphocyte predominance Hodgkin disease retain the molecular profile of germinal center B cells. In this paper, we investigated the expression in Hodgkin disease (45 cases and 3 cell lines) of 5 intracellular signaling molecules found in B cells. The Src family kinase Syk, the B-cell adaptor protein BLNK, and phospholipase C (PLC)-gamma2 were consistently absent from Reed-Sternberg cells, whereas 2 other Src kinases (Lyn and Fyn) were heterogeneously expressed in a proportion of cases (12% and 42%, respectively). In contrast, the tumor cells in all cases of lymphocyte predominance Hodgkin disease were positive for Fyn, Syk, BLNK, and PLC-gamma2, and Lyn immunostaining was seen in a minority of biopsies. These results indicate that in Reed-Sternberg cells, the defect in B-cell lineage marker expression includes a spectrum of molecules involved in intracellular signaling, a finding in keeping with recent gene expression profiling studies. Furthermore, the clear difference in expression of signaling proteins between the 2 major subtypes of Hodgkin disease may be of diagnostic value.

The prognostic value of histologic classification and single histomorphologic parameters in Hodgkin disease has been widely debated in the literature. Whereas several former studies identified single parameters to be of clinical relevance, some recent reports have doubted the prognostic value of histology using modern treatment. Grading of the largest histologic category of Hodgkin disease, nodular sclerosis (NS), has been controversially discussed concerning clinical relevance. In this study, 965 cases of NS were reviewed to assess 9 histomorphologic parameters. The histologic results were correlated with laboratory and clinical findings and with overall survival and disease-free survival. Based on these results, a new grading of the NS category was established. The new grading, based on the 3 criteria eosinophilia, lymphocyte depletion, and atypia of the Hodgkin/Reed-Sternberg cells, was a significant indicator of prognosis in intermediate and advanced stages. Patients investigated in this study represent an outstanding collection because all of them were enrolled in the prospective multicenter clinical trial of the German Hodgkin Lymphoma Study Group. All of them had been staged uniformly according to the Ann Arbor system and had received stage-adapted modern treatment according to multimodality protocols. A subtle analysis of histology could represent a possible way to identify patients with a significantly better or worse prognosis. This new grading should help to avoid overtreatment to reduce severe therapy-related side effects such as acute toxicity and chronic sequelae such as cardiopulmonary complications and secondary neoplasias.

Hodgkin and Reed-Sternberg (HRS) cells represent the malignant cells in classical Hodgkin lymphoma (HL). Because their immunophenotype cannot be attributed to any normal cell of the hematopoietic lineage, the origin of HRS cells has been controversially discussed, but molecular studies established their derivation from germinal center B cells. In this study, gene expression profiles generated by serial analysis of gene expression (SAGE) and DNA chip microarrays from HL cell lines were compared with those of normal B-cell subsets, focusing here on the expression of B-lineage markers. This analysis revealed decreased mRNA levels for nearly all established B-lineage-specific genes. For 9 of these genes, lack of protein expression was histochemically confirmed. Down-regulation of genes affected multiple components of signaling pathways active in B cells, including B-cell receptor (BCR) signaling. Because several genes down-regulated in HRS cells are positively regulated by the transcriptional activator Pax-5, which is expressed in most HRS cells, we studied HL cell lines for mutations in the Pax-5 gene. However, no mutations were found. We propose that the lost B-lineage identity in HRS cells may explain their survival without BCR expression and reflect a fundamental defect in maintaining the B-cell differentiation state in HRS cells, which is likely caused by a novel, yet unknown, pathogenic mechanism.

The derivation of follicular lymphomas (FLs) from germinal centers is not only supported by their morphologic appearance with a nodular growth pattern and a germinal center-like cellular composition, but also by the presence of ongoing somatic hypermutation (a germinal center B cell-specific process) during their clonal expansion. The intraclonal sequence diversity of the tumor cells and their follicular growth pattern allows one to analyze lymphoma cell dissemination and the way the tumor "metastasizes" to distinct follicles. In the present study, we analyzed individual follicles of 3 FLs by micromanipulation of single cells from individual lymphoma follicles and amplification of immunoglobulin V region genes. Genealogical trees for the V(H) and the V(L) gene rearrangements were constructed to analyze the clonal relationship among individual cells of 3 distinct follicles of each case. In all 3 cases there is evidence that distinct tumor follicles are founded by many tumor cells, suggesting that there is extensive migration of the tumor cells among follicles. The observation that the tumor cells of FLs retain their follicular growth patterns despite this cellular migration supports the idea that they depend on the follicular microenvironment for their clonal expansion.

Comparative genomic hybridization studies have shown gains in chromosome region 2p as the most common imbalance in classical Hodgkin lymphoma (cHL). The minimal region of gain contained 2 candidate oncogenes, REL and BCL11A. This study examined the involvement of REL and BCL11A loci in 44 primary cases of cHL by combined immunophenotyping and interphase cytogenetics (FICTION). A median 2p13 copy number above the tetraploid range was detected in 24 (55%) cases. Adjustment for centromere 2 copy number indicated gains of 2p13 in 11 of 31 cHLs (35%) with 8 (26%) high-level amplifications. One cHL displayed selective amplification of the REL locus not affecting BCL11A; another case studied by FICTION and a cHL with cytogenetic 2p change investigated by fluorescence in situ hybridization showed signal patterns suggesting breakpoints in the region spanned by the REL probe. These data indicate that REL rather than BCL11A may be the target of the 2p13 alterations in cHL.

Progressively transformed germinal centers (PTGCs) are histologic structures mainly composed of small resting B cells and intermingled proliferating centroblast-like cells. The B-cell differentiation processes within PTGCs and their relation to classical germinal centers (GC) and to lymphocyte-predominant Hodgkin disease (LPHD), with which PTGCs are often associated, are largely unknown. To address these issues, single small resting (Ki67-) and proliferating (Ki67+) centroblast-like cells were isolated from 7 PTGCs of 5 lymph nodes, and rearranged immunoglobulin genes were amplified and sequenced. Most small resting B cells were clonally unrelated, and most carried unmutated immunoglobulin gene rearrangements resembling mantle zone B cells. Small resting B cells with mutated immunoglobulin gene rearrangements may represent centrocytes, memory B cells, or both. Among the centroblast-like Ki67+ cells, expanded B-cell clones were observed in 6 of 7 PTGCs analyzed. Clonally related V region genes showed extensive intraclonal diversity, and the mutation pattern indicated stringent selection of the cells for the expression of functional antigen receptors. Thus, somatic hypermutation, clonal expansion, and selection occur also in the disorganized PTGC microenvironment, as in classical GCs. In lymph nodes affected by PTGCs, no clonal expansion across the borders of individual PTGCs was observed, distinguishing PTGCs from LPHD.

In most cases, Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (HD) carry rearranged immunoglobulin (Ig) genes and thus derive from B cells. In rare cases, HRS cells originate from T cells. However, based on the unusual immunophenotype of HRS cells, often showing coexpression of markers typical for different hematopoietic lineages, and the regular detection of numerical chromosomal abnormalities, it has been speculated that HRS cells might represent cell fusions. Five cases of HD with 2 rearranged IgH alleles were analyzed for the presence of additional IgH alleles in germline configuration as a potential footprint of a cell fusion between a B and a non-B cell. Similarly, one case of T-cell-derived HD with biallelic T-cell receptor beta (TCRbeta) rearrangements was studied for the presence of unrearranged TCRbeta alleles. In none of the 6 cases was evidence for additional IgH (or TCRbeta) alleles obtained, strongly arguing against a role of cell fusion in HRS cell generation.

Epstein-Barr virus (EBV) can be detected in the tumor cells of approximately 40% of cases of classical Hodgkin disease (cHD). Clonality studies suggest that infection of the neoplastic Hodgkin and Reed/Sternberg (HRS) cells occurs before tumor clone expansion. In EBV-positive cases, variable numbers of EBER-positive small B cells are sometimes also observed that immunohistologically differ from the neoplastic cells by lack of CD30 and latent membrane protein 1 expression. To analyze the clonal relationship between these EBV(+) cells and the HRS cells, single EBV-infected CD30(-) B cells, as well as HRS cells from 3 cases of EBV-positive cHD were micromanipulated, their immunoglobulin gene rearrangements amplified and then compared with each other. In 2 cases, all small EBV-infected cells were clonally unrelated to the HRS cells. In a third case, 2 of 29 small CD30(-) cells were found to carry HRS cell-specific rearrangements. Thus, small CD30(-) EBV-infected B cells in cHD belong to the HRS tumor clone rarely, if at all. In all cases, small clones unrelated to the HRS cell clones were identified among the small EBV(+) CD30(-) cells. The vast majority of small EBV(+) CD30(-) B cells was found to carry somatically mutated V region genes, indicating that in lymph nodes of patients with HD, like in the peripheral blood of healthy individuals, EBV persists in memory B cells.

Paraffin blocks and clinical data from 521 patients with lymphocyte predominance Hodgkin disease (LPHD) diagnosed between 1970 and 1994 were collected from 16 European and United States oncological centers to establish the pathologic and clinical characteristics of a large patient cohort, to determine how frequent T-cell-rich large B-cell lymphoma (TCRLBCL) is among LPHD, and to find differential diagnostic criteria distinguishing between the 2 lymphoma categories. For this purpose, conventionally and immunohistologically stained sections were reviewed by a panel of hematopathologists. The diagnosis of LPHD was confirmed in only 219 of the 388 assessable cases (56.5%). This low confirmation rate was due mainly to the presence of a new variant of classical Hodgkin disease (CHD), which resembled, in terms of nodular growth and lymphocyte-richness, nodular LPHD and, in terms of the immunophenotype of the tumor cells, CHD and was designated nodular lymphocyte-rich CHD (NLRCHD). The nodules of LRCHD consisted-as in nodular LPHD-predominantly of B cells but differed from those present in LPHD in that they represented expanded mantle zones with atrophic germinal centers. Clinically, patients with LPHD and NLRCHD showed similar disease characteristics at presentation but differed in the frequency of multiple relapses and prognosis after relapse. Patients with LPHD and NLRCHD clearly differed from patients with CHD with nodular sclerosis or mixed cellularity, as they presented with an earlier disease stage and infrequent mediastinal involvement. As 97% of the LPHD cases showed a complete or partial nodular growth pattern, their differentiation from TCRLBCL was a rare problem in the present series. (Blood. 2000;96:1889-1899)

Although eosinophilic granulocytes are frequently observed in lymphatic tissue of Hodgkin's patients, no substantial data reveal the prognostic role, if any, of tissue eosinophilia. Thus, eosinophilia was analyzed histologically in 1511 diagnostic biopsy specimens of patients treated under protocol therapy of the German Hodgkin's Lymphoma Study Group between 1988 and 1994. Prominent eosinophilia was seen in 38% of cases, which differed among the histologic types of Hodgkin's disease (HD): none in lymphocyte predominant, 14% in lymphocyte rich classical, 40% in nodular sclerosis grade 1 (NS-1), 55% in nodular sclerosis grade 2, 43% in mixed cellularity (MC), and 54% in lymphocyte depleted. In a multivariate analysis, tissue eosinophilia proved to be the strongest prognostic factor for freedom from treatment failure (P <. 001) and overall survival (P <.001) in a stage-stratified model. Among NS-1 patients, the effect was highly significant. In MC, no significant effect of eosinophilia on survival could be demonstrated. Eosinophils secrete CD30 ligand that is capable of binding to CD30 positive HD cells. The activation of TRAF2, followed by NF-kappaB, which occurs on CD30L/CD30 binding, may explain the neoplastic proliferation and apoptosis protection of HD cells. TRAF2 is also activated by EBV-LMP expression, which is detectable in the majority of MC but not NS cases. In addition to the possibility that eosinophils are only passive indicators for other unknown prognostic determinants, it may be concluded that the positive clinical outcome of eosinophilia-negative NS cases could be due to lower NF-kappaB activity. (Blood. 2000;95:1207-1213)

In rare cases of B-cell chronic lymphocytic leukemia (B-CLL), large cells morphologically similar to or indistinguishable from Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin's disease (HD) can be found in a background of otherwise typical B-CLL. To test these HRS-like cells for a potential clonal relationship to the B-CLL cells, single cells were micromanipulated from immunostained tissue sections, and rearranged immunoglobulin genes were amplified from HRS-like cells and B-CLL cells and sequenced. The same variable (V) gene rearrangements with shared and distinct somatic mutations were found in HRS-like and B-CLL cells from 1 patient, which indicates derivation of these cells from 2 distinct members of a germinal-center B-cell clone. Separate clonal V gene rearrangements were amplified from HRS-like and B-CLL cells from 2 other patients, showing concomitant presence of 2 distinct expanded B-cell clones. Epstein-Barr virus (EBV) was detected in the HRS-like cells of these 2 latter cases, indicating clonal expansion of an EBV-harboring B cell in the setting of B-CLL. There is evidence that HRS-like cells in B-CLL, like HRS cells in HD, derive from germinal-center B cells. In all cases, somatic mutations have been detected in the rearranged V genes of the HRS-like cells, and in 1 of the EBV-positive HRS-like cell clones, somatic mutations rendered an originally functional V gene rearrangement nonfunctional. We speculate that the HRS-like cells in B-CLL represent potential precursors for HRS cells causing HD.

Point mutations of the p53 tumor suppressor gene are a frequent finding in human carcinomas and are thought to be an important oncogenic event. In non-Hodgkin lymphomas, p53 mutations occur in a minor fraction of cases. However, conclusive data are still lacking for Hodgkin's disease (HD) where the analysis meets technical problems. The neoplastic tumor cell clone in HD is represented by the large Hodgkin and Reed-Sternberg (HRS) cells, which account for only a minority of all cells in the tumor tissue (often <1%). To identify putative HRS cell-specific mutations, single HRS cells were micromanipulated from frozen tissue sections of HD biopsy specimens. Exons 4 to 8 of the p53 gene (in which more than 90% of p53 mutations associated with human neoplasms occur) were amplified from these single cells and sequenced. Mutations of p53 were not found in HRS cells of any of 8 cases of HD analyzed. We conclude that mutation of the p53 gene is only rarely, if at all, involved in the pathogenesis of HD.

T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression after acquiring a crippling mutation within the initially functional kappa light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJH rearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.

Hodgkin's disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VH gene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VH rearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 10(5) mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34(+) stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation., (Copyright 1998 by The American Society of Hematology.)

The anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, was used in a phase I dose escalation trial in patients with refractory Hodgkin's lymphoma. The IT was constructed by linking the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin-A. All patients in this trial were heavily pretreated with a mean of 5 (range, 2 to 8) different prior therapies, including autologous bone marrow transplantation in 8 of 15. The mean age was 29 years (range, 19 to 34 years). Thirteen of 15 patients had advanced disease (stage IV) with massive tumor burdens and 6 of 15 had B symptoms. The IT was administered intravenously over 4 hours on days 1, 3, 5, and 7 for total doses per cycle of 5, 10, 15, or 20 mg/m2. Patients received one to four cycles of treatment. The peak serum concentration of intact IT varied from 0.2 to 9.7 micrograms/mL. The serum half life (T1/2) of the IT ranged from 4.0 to 10.5 hours (mean, 6.1 hours). Side effects were related to vascular leak syndrome (VLS), ie, decreases in serum albumin, edema, weight gain, hypotension, tachycardia, myalgia, and weakness. Two patients had a National Cancer Institute (NCI) grade 2 allergic reaction with generalized urticaria and mild bronchospasm. At 15 mg/m2, 1 patient experienced a grade 3 myalgia. All 3 patients receiving 20 mg/m2 experienced NCI grade 3 toxicities (edema, nausea, dyspnea or tachycardia) and 1 patient had NCI grade 4 myalgia. Thus, the maximal tolerated dose was 15 mg/m2. Seven of 15 patients made human antiricin antibodies (> or = 1.0 microgram/mL) and 6 of 15 developed human antimouse antibodies (> or = 1.0 microgram/mL). Clinical response included 2 partial remissions, 1 minor response, 3 stable diseases, and 9 progressive diseases. As has been predicted from the preclinical tests, these data seem to indicate clinical efficacy of this new IT in heavily pretreated Hodgkin's patients, thus warranting further clinical investigation.