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Purpose: Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene.Experimental Design: To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon.Results: Using array-based comparative genomic hybridization analysis, we detected a region of ∼1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser194 phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser194 phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest.Conclusion: Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit from Taxol-based chemoradiotherapy.

Supplementary Materials and Methods; Supplementary Table S1.

Supplementary Table S1 from Mechanisms and Effects of Loss of Human Leukocyte Antigen Class II Expression in Immune-Privileged Site-Associated B-Cell Lymphoma

Purpose and Experimental Design: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR and larger hemizygous deletions of the MHC region. To better understand the significance of down-regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we analyzed global gene expression patterns in a series of 26 testicular DLBCL after characterization of these deletions.Results: Low levels of HLA-DR mRNA in whole testicular DLBCL samples were associated with a strong down-regulation of numerous immune-related genes specific for T cells, macrophages, antigen presentation and processing, lymphocyte activation, chemokines and chemokine receptors, and the complement system. The number of CD3+ tumor-infiltrating T cells was also significantly lower in low expressors of HLA-DR mRNA. Interestingly, hemizygous and homozygous deletions in the MHC region did not have any additional effect on global gene expression.Conclusion: In conclusion, we found that loss of HLA class II mRNA expression in testicular DLBCL is associated with a significant change in global gene expression patterns. This effect is independent of the mechanism causing the down-regulation of HLA class II genes in the lymphoma cells.

The immune system is composed of two closely collaborative systems, an innate and an adaptive system. The innate immune system is a constitutive present system that can act rapidly to eradicate microbes. The primary cells of the innate immune system are macrophages, granulocytes, natural killer cells, and dendritic cells. The adaptive system can be divided in a humoral and cellular response. The humoral response is characterized by activation of B lymphocytes with subsequent maturation into plasma cells and production of antibodies, whereas a cellular immune response is characterized by transformation of T lymphocytes into cytotoxic T cells, capable of killing virally infected cells.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.

AimsMethods and resultsMost validation studies on digital pathology diagnostics have been performed in single institutes. Because rapid consultation on cases with extramural experts is one of the most important uses for digital pathology laboratory networks, the aim of this study was to validate a whole-slide image-based teleconsultation network between three independent laboratories.Each laboratory contributed 30 biopsies and/or excisions, totalling 90 specimens (776 slides) of varying difficulty and covering a wide variety of organs and subspecialties. All slides were scanned centrally at x40 scanning magnification and uploaded, and subsequently assessed digitally by 16 pathologists using the same image management system and viewer. Each laboratory was excluded from digital assessment of their own cases. Concordance rates between the two diagnostic modalities (light microscopic versus digital) were compared. Loading speed of the images, zooming latency and focus quality were scored. Leaving out eight minor discrepancies without any clinical significance, the concordance rate between remote digital and original microscopic diagnoses was 97.8%. The two cases with a major discordance (for which the light microscopic diagnoses were deemed to be the better ones) resulted from a different interpretation of diagnostic criteria in one case and an image quality issue in the other case. Average scores for loading speed of the images, zooming latency and focus quality were 2.37 (on a scale up to 3), 2.39 (scale up to 3) and 3.06 (scale up to 4), respectively.ConclusionsThis validation study demonstrates the suitability of a teleconsultation network for remote digital consultation using whole-slide images. Such networks may contribute to faster revision and consultation in pathology while maintaining diagnostic standards.

As an immune escape mechanism tumor cells may downregulate expression of Human Leukocyte Antigens (HLA). Loss of HLA class I and class II has been described in various subtypes of diffuse large B cell lymphoma (DLBCL), including the not otherwise specified (NOS) subgroup. 1,2 We analyzed HLA class I and class II expression in DLBCL not otherwise specified (NOS) to investigate whether there is an association between HLA expression, cell of origin (COO) and the recently reported FOXP1 expression in non-GCB DLBCL. 3 This article is protected by copyright. All rights reserved.

Current genomic models in diffuse large B-cell lymphoma (DLBCL) are based on single tumor biopsies, which might underestimate heterogeneity. Data on mutational evolution largely remains unknown. An exploratory study using whole exome sequencing on paired (primary and relapse) formalin fixed paraffin embedded DLBCL biopsies (n = 14) of 6 patients was performed to globally assess the mutational evolution and to identify gene mutations specific for relapse samples from patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. A minority of the mutations detected in the primary sample (median 7.6%, range 4.8&ndash, 66.2%) could not be detected in the matching relapse sample. Relapsed DLBCL samples showed a mild increase of mutations (median 12.5%, range 9.4&ndash, 87.6%) as compared to primary tumor biopsies. We identified 264 genes possibly related to therapy resistance, including tyrosine kinases (n = 18), (transmembrane) glycoproteins (n = 73), and genes involved in the JAK-STAT pathway (n = 7). Among the potentially resistance related genes were PIM1, SOCS1, and MYC, which have been reported to convey a risk for treatment failure. In conclusion, we show modest temporal heterogeneity between paired tumor samples with the acquisition of new mutations and identification of genes possibly related to therapy resistance. The mutational evolution could have implications for treatment decisions and development of novel targeted drugs.

Author(s): Ustun, Celalettin; Morgan, Elizabeth; Moodie, Erica EM; Pullarkat, Sheeja; Yeung, Cecilia; Broesby-Olsen, Sigurd; Ohgami, Robert; Kim, Young; Sperr, Wolfgang; Vestergaard, Hanne; Chen, Dong; Kluin, Philip M; Dolan, Michelle; Mrozek, Krzysztof; Czuchlewski, David; Horny, Hans-Peter; George, Tracy I; Kristensen, Thomas Kielsgaard; Ku, Nam K; Yi, Cecilia Arana; Moller, Michael Boe; Marcucci, Guido; Baughn, Linda; Schiefer, Ana-Iris; Hilberink, JR; Pullarkat, Vinod; Shanley, Ryan; Kohlschmidt, Jessica; Coulombe, Janie; Salhotra, Amandeep; Soma, Lori; Cho, Christina; Linden, Michael A; Akin, Cem; Gotlib, Jason; Hoermann, Gregor; Hornick, Jason; Nakamura, Ryo; Deeg, Joachim; Bloomfield, Clara D; Weisdorf, Daniel; Litzow, Mark R; Valent, Peter; Huls, Gerwin; Perales, Miguel-Angel; Borthakur, Gautam | Abstract: BackgroundAlthough the prognosis of core-binding factor (CBF) acute myeloid leukemia (AML) is better than other subtypes of AML, 30% of patients still relapse and may require allogeneic hematopoietic cell transplantation (alloHCT). However, there is no validated widely accepted scoring system to predict patient subsets with higher risk of relapse.MethodsEleven centers in the US and Europe evaluated 247 patients with t(8;21)(q22;q22).ResultsComplete remission (CR) rate was high (92.7%), yet relapse occurred in 27.1% of patients. A total of 24.7% of patients received alloHCT. The median disease-free (DFS) and overall (OS) survival were 20.8 and 31.2 months, respectively. Age, KIT D816V mutated (11.3%) or nontested (36.4%) compared with KIT D816V wild type (52.5%), high white blood cell counts (WBC), and pseudodiploidy compared with hyper- or hypodiploidy were included in a scoring system (named I-CBFit). DFS rate at 2 years was 76% for patients with a low-risk I-CBFit score compared with 36% for those with a high-risk I-CBFit score (P l 0.0001). Low- vs high-risk OS at 2 years was 89% vs 51% (P l 0.0001).ConclusionsI-CBFit composed of readily available risk factors can be useful to tailor the therapy of patients, especially for whom alloHCT is not need in CR1 (ie, patients with a low-risk I-CBFit score).

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.

BackgroundAimsThe benefits of digital pathology for workflow improvement and thereby cost savings in pathology, at least partly outweighing investment costs, are being increasingly recognised. Successful implementations in a variety of scenarios have started to demonstrate the cost benefits of digital pathology for both research and routine diagnosis, contributing to a sound business case encouraging further adoption. To further support new adopters, there is still a need for detailed assessment of the impact that this technology has on the relevant pathology workflows, with an emphasis on time-saving.To assess the impact of digital pathology adoption on logistic laboratory tasks (i.e. not including pathologists' time for diagnosis-making) in the Laboratorium Pathologie Oost Nederland, a large regional pathology laboratory in The Netherlands.Methods and resultsConclusionsTo quantify the benefits of digitisation, we analysed the differences between the traditional analogue and new digital workflows, carried out detailed measurements of all relevant steps in key analogue and digital processes, and compared the time spent. We modelled and assessed the logistic savings in five workflows: (i) routine diagnosis; (ii) multidisciplinary meeting; (iii) external revision requests; (iv) extra stainings; and (v) external consultation. On average, >19 working hours were saved on a typical day by working digitally, with the highest savings in routine diagnosis and multidisciplinary meeting workflows.By working digitally, a significant amount of time could be saved in a large regional pathology laboratory with a typical case mix. We also present the data in each workflow per task and concrete logistic steps to allow extrapolation to the context and case mix of other laboratories.

CD30 is a transmembrane receptor, normally not expressed by mast cells, which regulates proliferation/apoptosis and antibody responses. Aberrant expression of CD30 by mastocytosis mast cells and interaction with its ligand CD30L (CD153) appears to play an important role in the pathogenesis and clinical presentation of systemic mastocytosis. This article highlights the expression profile and role of CD30 and CD3OL in physiologic and pathologic conditions, the applicability of CD30 as a marker for systemic mastocytosis, the consequences of mast cell-expressed CD30, and the possibility of future anti-CD30 based cytoreductive therapies.

Objectives/Hypothesis: To examine the prognostic value of hypoxia inducible factor HIF-1a, CA-IX, and OPN on clinical outcome in patients with T1-T2 supraglottic laryngeal squamous cell carcinoma (LSCC) treated with primarily radiotherapy (RT). Study Design: Retrospective cohort study. Methods: Tumor tissue sections of 60 patients with T1-T2 supraglottic LSCC treated with primarily radiotherapy were assessed immunohistochemically for expression of HIF-1a, CA-IX, and OPN. The relationship of protein expression and classical clinical parameters with clinical outcome was studied, using Cox regression and Kaplan-Meier survival analyses. Results: Neither HIF-1a nor CA-IX was of prognostic significance toward local control or overall survival in T1-T2 supraglottic LSCC. Cox regression survival analysis showed no relation between HIF-1a or CA-IX expression and local control (HR [hazard ratio] 1.07, CI [95% confidence interval] 0.29-3.87; HR 0.34, CI 0.04-2.58). Furthermore, OPN expression was not associated with local control (HR 1.37, CI 0.45-4.17) and overall survival (HR 0.99, CI 0.44-2.21). Our earlier findings in T1-T2 glottic LSCC (Schrijvers et al., 2008) could not be confirmed. Conclusion: The absence of prognostic significance for HIF-1a and CA-IX toward local control in supraglottic LSCC, unlike glottic LSCC, suggests that supraglottic LSCC might represent another biological entity.

Risk indicators of indolent systemic mastocytosis (ISM) in adults with clinical suspicion of ISM without accompanying skin lesions [urticaria pigmentosa (UP)] are lacking. This study aimed at creating a decision tree using clinical characteristics, serum tryptase, and the urinary histamine metabolites methylimidazole acetic acid (MIMA) and methylhistamine (MH) to select patients for bone marrow investigations to diagnose ISM.Retrospective data analysis of all adults, in whom bone marrow investigations were performed to diagnose ISM, was carried out.In total, 142 patients were included. SM was absent in all 44 patients with tryptase10 μg/l, in 45 of 98 (46%) patients with tryptase ≥10 μg/l and in 18 of 52 patients (35%) with tryptase20 μg/l. Above 43 μg/l, all patients had ISM (n = 11). Male gender, insect venom anaphylaxis as presenting symptom, tryptase, MIMA, and MH were independent ISM predictors. If tryptase was ≥10 μg/l, the diagnostic accuracy of MIMA and MH was high (areas under the ROC curve 0.92).In suspected patients without UP, the ISM risk is very low (if present at all) if tryptase is10 μg/l. If tryptase is ≥10 μg/l, this risk depends on MIMA and MH, being low if these are normal, but high if these are elevated. Male gender and insect venom anaphylaxis are additional risk indicators. We recommend refraining from bone marrow examinations in suspected patients without UP if tryptase is10 μg/l. Our results question the reliability of the minor diagnostic World Health Organization criterion of tryptase20 μg/l.

Contains fulltext : 80422.pdf (Publisher’s version ) (Closed access) Mantle cell lymphoma is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear whether the mutational status is stable over time within a given case, whether hypermutation might be influenced by therapy and how it is related to other relevant biological features of mantle cell lymphoma. In this study, we analyzed 23 typical mantle cell lymphoma cases with respect to mutational status and compared the results with clinicopathological and genetic data to determine whether the presence of mutation indicates a subentity with clinical or pathological relevance. We found somatic hypermutation in 26% of our cases and, interestingly, one case showed ongoing somatic hypermutation. In tumor cells of both mutated and unmutated cases, we found a preferential usage of V(H)3-21 (23%) and V(H)4-34 (19%). No significant correlations were found between mutation status and the other morphological and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status in mantle cell lymphoma is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance.

Purpose In the new WHO–European Organisation for Research and Treatment of Cancer (WHO-EORTC) classification for cutaneous lymphomas three major groups of primary cutaneous B-cell lymphoma (CBCL) are distinguished: primary cutaneous marginal zone B-cell lymphoma (PCMZL) and primary cutaneous follicle center lymphoma (PCFCL) with a good prognosis, and primary cutaneous large B-cell lymphoma, leg type (PCLBCL-LT), with an intermediate-level prognosis. This study aimed to assess the clinical significance of the new classification compared with previous classification schemes (EORTC 1997; WHO 2001) and to define prognostic factors within the newly defined categories. Patients and Methods In the present study clinical data and histologic sections of 300 patients with CBCL, formerly classified according to the EORTC classification, were reviewed and reclassified according to the WHO and the new WHO-EORTC classification schemes. Results After reclassification, the study comprised 71 patients with PCMZL, 171 patients with PCFCL, and 58 patients with PCLBCL-LT, showing 5-year disease-specific survivals of 98%, 95%, and 50%, respectively. When compared with the EORTC and WHO schemes, 5.3% and 36.3% of patients with CBCL were reclassified into another prognostic category. Multivariate analysis of PCFCL revealed localization on the leg and expression of FOXP1 as independent parameters associated with a poor prognosis. Expression of Bcl-2 or MUM-1 had no significant effect on survival in this group. In PCLBCL-LT, no independent prognostic parameters were found. Conclusion These results emphasize the clinical significance of the WHO-EORTC classification, but suggest that within the group of PCFCL, distinction should be made between lymphomas presenting on the legs and lymphomas presenting at other sites.

Background:The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma.Methods:Tumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation.Results:A classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappa B target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens.Conclusions:Gene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma.

Chromosomal translocations involving the immunoglobulin (Ig) receptor loci usually disrupt and silence these loci. On the basis of observations in follicular lymphoma (FL) with downstream Ig heavy chain (IGH) class switch recombination (CSR), we hypothesized that downstream CSR-mediated chromosomal translocations would leave the V(D)J-Cmu transcription unit intact, thereby still allowing IgM expression from the IGH allele involved in the translocation. To test this hypothesis, we analyzed biallelic IGH translocations in the IgM-expressing cell line Z-138 by interphase FISH, DNA fiber-FISH, long-distance vectorette PCR, and DNA sequencing. One IGH allele was involved in a t(11;14), showing a break in the JH region that juxtaposed the El,L enhancer and the 3' Calpha enhancers to the cyclin D1 gene. The other IGH allele contained a t(8; 14) breakpoint involving the 3' end of a Sgamma region, whereas the reciprocal breakpoint at 8q24 was approximately 40 kb centromeric of MYC. Molecular analysis showed that this IGH allele harbored a normal V(D)J-Cmu complex, which is responsible for IgM expression. These data show that chromosomal breakpoints such as the t(8; 14) can occur in downstream IGH constant regions and do not necessarily interfere with Ig expression. (C) 2004 Wiley-Liss, Inc.

Contains fulltext : 47779.pdf (Publisher’s version ) (Closed access) Follicular lymphoma (FL) is a disease characterized by a long clinical course marked by frequent relapses that vary in clinical aggressiveness over time. Therefore, the main dilemma at each relapse is the choice for the most effective treatment for optimal disease control and failure-free survival while at the same time avoiding overtreatment and harmful side effects. The selection for more aggressive treatment is currently based on histologic grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using supervised classification on a training set of paired samples from patients who experienced either an indolent or aggressive disease course, a gene expression profile of 81 genes was established that could, with an accuracy of 100%, distinguish low-grade from high-grade disease. This profile accurately classified 93% of the FL samples in an independent validation set. Most important, in a third series of FL cases where histologic grading was ambiguous, precluding meaningful morphologic guidance, the 81-gene profile shows a classification accuracy of 94%. The FL stratification profile is a more reliable marker of clinical behavior than the currently used histologic grading and clinical criteria and may provide an important alternative to guide the choice of therapy in patients with FL both at presentation and at relapse.

Translocations involving band 3q27, affecting the major breakpoint region (MBR) of BCL6, are common in diffuse large B-cell lymphomas (DLBCLs). Recent data suggest an alternative breakpoint cluster region (ABR) located between 245 and 285 kb 5' of BCL6, which might be associated with Follicular Lymphoma (FL). Ten DLBCLs and 9 FLs grade 3B with cytogenetic rearrangements at 3q27 were studied by fluorescence in situ hybridization (FISH) to discriminate between breakpoints at the ABR and MBR. Eight DLBCLs contained a breakpoint in the MBR, and 6 FL grade 3B (FL3B) cases contained a breakpoint in the ABR. No specific chromosomal partners could be identified in both groups. Previously published data have suggested that FL3B cases with 3q27 aberrations are closely related to the majority of DLBCLs of germinal center cell origin. However, our findings suggest that the mechanism of 3q27 rearrangement in FL3B cases is similar to the mechanism in follicular lymphomas grade 1,2, and 3A cases. (c) 2005 Wiley-Liss, Inc.

The t(9;11) has been described in patients with acute myeloid leukemia (AML), and two genes [AF9 (at 9p21) and FBP17 (at 9q34)] have been cloned as fusion partners of the MLL gene. From an AML-M5 with a t(9;11)(q34;q23), we identified a novel MLL fusion partner, AF9Q34. The AF9Q34 protein shows high homology with nGAP, a RAS GTPase-activating protein (RASGAP), and contains the highly conserved GRD and FLR motifs characteristic of RASGAPs. Recently, the rat homologue (DAB2IP) also was identified and reported to act as a RASGAP both in vivo and in vitro. RASGAPs negatively regulate the activity of RAS proteins that modulate diverse cellular processes by cycling between an inactive GDP-bound and an active GTP-bound state. In addition, the NH2 terminus harbors an amino acid stretch with homology to the pleckstrin homology (PH) domain implicated in regulating the interaction between RAS and the catalytic domain of RASGAP. As a result of the breakpoint in the AF9Q34–MLL fusion protein, this PH domain is disrupted. This suggests that because of the translocation, the normal function of the AF9Q34 gene is aborted. Thus, AF9Q34 encodes a novel RASGAP gene that appears to be deregulated as a result of the translocation. The identification of this RASGAP protein in a novel MLL fusion implies that an indirect RAS-deregulating mechanism could be involved in leukemic transformation. © 2004 Wiley-Liss, Inc.

Item does not contain fulltext Minor histocompatibility antigens can induce cytotoxic T cells that play an important role in the graft-versus-leukemia and graft-versus-host-disease (GvHD) activity after stem cell transplantation. Minor histocompatibility antigens (mHags) with expression limited to the hematopoietic system may have a prominent role in the graft-versus-leukemia reaction. Earlier in vitro studies demonstrated that cytotoxic T cells specific for the minor histocompatibility antigen HA-1 only lysed cells of hematopoietic origin. Despite this limited expression, an HA-1 mismatch is associated with GvHD. Yet, the hematopoietic-restricted HA-1 membrane expression motivated us to develop an ex vivo HA-1-specific protocol for cellular immunotherapy of relapsed leukemia. To ensure the feasibility and safety of such cellular therapy, broad HA-1 RNA analysis is indispensable. Here we demonstrate the hematopoietic-restricted expression at the HA-1 gene transcriptional level with high RNA expression in normal and in malignant hematopoietic cells and background expression levels in nonhematopoietic cells. In tissues that showed low HA-1 RNA expression, hematopoietic cells were present as demonstrated by CD45 RNA expression analyzed in parallel. Thus, the mHag HA-1 can function as an excellent target antigen for immunotherapy of hematological malignancies with no or low risk of GvHD.

To the Editor: We report the case of an elderly male referred with pulmonary opacities and extensive mediastinal lymphadenopathy suspicious for lung cancer. He was found to have Rosai–Dorfman disease in a lymph node and concomitant immunoglobulin (Ig)G4-related disease in the lung and kidney. An 83-yr-old male was referred with a pulmonary consolidation in the left lung with extensive lymphadenopathy without a pathological diagnosis. His past medical record showed gout for which he used colchicine only occasionally. Initially, he was referred to an internist with complaints of loss of appetite, weight loss and fatigue. His sense of taste was diminished and he had a dry mouth. No dyspnoea was present. He had a longstanding, non-productive cough. Until recently, he was remarkably fit for his age. He had quit smoking 45 yrs earlier after 30 pack-yrs. Physical examination was unremarkable except for a firm lymph node in the left supraclavicular region and minor pulmonary crackles basally on both sides. Spirometry results were normal. Initial laboratory analysis showed a normal complete blood count, and slightly elevated creatinine (168 μmol·L−1) and C-reactive protein (35 mg·L−1). A chest radiograph showed a hazy consolidation radiating from the left hilum to the periphery of the lung suggesting lung cancer. On computed tomography, diffuse opacification in the left lower lobe was seen with concomitant hilar, mediastinal (paratracheal and subcranial) and left supraclavicular lymphadenopathy. Fibreoptic bronchoscopy in our institution again revealed no endobronchial lesions. Pathological examination of lavage fluid from the left lower …

Loss of heterozygosity (LOH) is a major mechanism for inactivation of tumor-suppressor genes and has been observed in various solid tumors and lymphomas, The human leukocyte antigen (HLA) region is located at chromosome band 6p21.3, and loss or alteration of this region may provide tumor cells with a mechanism to escape from the immune system. We previously identified small homozygous deletions within the HLA class 11 region in many of the diffuse large B-cell lymphomas (DLCLs) of the central nervous system (CNS) and the testis. In the present study, we focused on the mechanism leading to LOH in the HLA region. Twenty microsatellite markers, of which 12 were specific for HLA, were applied on 11 extranodal DLCLs of the CNS and 28 of the testis. Additionally, fluorescence in situ hybridization with seven HLA-specific probes and a centromere 6-specific probe was performed on 20 cases to study the mechanism of LOH. In contrast to previously published data on spontaneously mutated lymphoblastoid cell lines, intrachromosomal hemizygous deletion, not mitotic recombination, was the major cause of LOH of the HLA region in these lymphomas. However, opposed to data in colorectal cancer. these deletions were rarely (one of nine cases) associated with an interchromosomal rearrangement such as a translocation. (C) 2002 Wiley-Liss, Inc.

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal VH gene diversity. VH-Cμ and VH-Cγ gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

The majority of anaplastic large cell lymphomas (ALCL) are associated with chromosomal abnormalities affecting the anaplastic lymphoma kinase (ALK) gene which result in the expression of hybrid ALK fusion proteins in the tumor cells. In most of these tumors, the hybrid gene comprises the 5' region of nucleophosmin (NPM) fused in frame to the 3' portion of ALK, resulting in the expression of the chimeric oncogenic tyrosine kinase NPM-ALK. However, other variant rearrangements have been described in which ALK fuses to a partner other than NPM. Here we have identified the moesin (MSN) gene at Xq11-12 as a new partner of ALK in a case of ALCL which exhibited a distinctive membrane-restricted pattern of ALK labeling. The hybrid MSN-ALK protein had a molecular weight of 125 kd and contained an active tyrosine kinase domain. The unique membrane staining pattern of ALK is presumed to reflect association of moesin with cell membrane proteins. In contrast to other translocations involving the ALK gene, the ALK breakpoint in this case occurred within the exonic sequence coding for the juxtamembrane portion of ALK. Identification of the genomic breakpoint confirmed the in-frame fusion of the whole MSN intron 10 to a 17 bp shorter juxtamembrane exon of ALK. The breakpoint in der(2) chromosome showed a deletion, including 30 bp of ALK and 36 bp of MSN genes. These findings indicate that MSN may act as an alternative fusion partner for activation of ALK in ALCL and provide further evidence that oncogenic activation of ALK may occur at different intracellular locations.

Using DNA fiber fluorescence in-situ hybridization (FISH) and 3-color interphase FISH, 2 cases of follicular lymphoma were identified in which the BCL2 gene was excised from 18q21 and inserted into the immunoglobulin heavy chain (IGH) locus at 14q32. Both the insertion breakpoint at 14q32 and the deletion breakpoint at 18q21 were cloned using inverse polymerase chain reaction. Sequence analysis showed that the JH sequences were juxtaposed to the 5′-side of BCL2, and the DH sequences were juxtaposed to the 3′-side of BCL2. There were breakpoints at both the JH and DH recombination signal sequences, and N-nucleotides were present at all breakpoint junctions. At theBCL2 locus, the 3′-breakpoints in both cases were localized at exactly the same nucleotide position, 6.2 kilobase downstream of the major breakpoint region, directly adjacent to a complete cryptic recombination signal sequence (RSS) consisting of a heptamer, a nonamer, and a 23–base pair (bp) spacer. The BCL25′-breakpoints were approximately 600 bp upstream of the gene, within the CA repeats. Although less evident than for the BCL23′-breakpoints, cryptic RSSs were also identified at these breakpoints, with a 12-bp spacer. On the basis of structural characteristics of these rearrangements, a model is proposed in which the BCL2 gene is deleted from its locus by recombination activation gene-1/-2 (RAG-1/-2)–mediated excision. The gene is subsequently inserted into the recombiningIGH locus, a process involving the formation of hybrid joints between the IGH coding ends and theBCL2 signal ends.

The expansion of follicular lymphomas (FLs) resembles, both morphologically and functionally, normal germinal center B-cell growth. The tumor cells proliferate in networks of follicular dendritic cells and are believed to be capable of somatic hypermutation and isotype switching. To investigate the relation between somatic mutation and heavy chain isotype expression, we analyzed the variable heavy (V(H)) chain genes of 30 FL samples of different isotypes. The V(H) genes of the FLs were heavily mutated (29.3 mutations on average). In addition, isotype-switched lymphomas contained more somatic mutations than immunoglobulin M-positive lymphomas (33.8 mutations per V(H) gene versus 23.0, respectively). In all but one of the FLs, the ratios of replacement versus silent mutations in the framework regions were low, independent of the absolute number of somatic mutations and the level of intraclonal variation. Analysis of relapse samples of 4 FLs showed no obvious increase in somatic mutation load in most FLs and a decrease in intraclonal variation in time. In 3 of 4 cases, we obtained evidence for selection of certain subclones, rather than clonal evolution. Our findings question if intraclonal variation is always a reflection of ongoing somatic hypermutation. This may have implications for the concept of antigen-driven lymphomagenesis. (Blood. 2000;95:2922-2929)

BACKGROUND There is no consistent definition at what age patients with non-Hodgkin lymphoma (NHL) are considered “elderly.” This might hamper well balanced decisions with respect to treatment. METHODS From a population-based NHL registry the age groups younger than 60 years, 60–64 years, 65–69 years, 70–74 years, and 75 years and older were analyzed in relation to the revised European-American lymphoma classification and to the age-adjusted International Prognostic Index (IPI). The prognostic value of the variables from the age-adjusted IPI was determined. The relative survival probabilities were calculated. RESULTS The incidence of diffuse large B-cell lymphoma (DLBL) increased with advancing age, as was the case for small lymphocytic lymphomas. Follicular lymphomas were less frequently encountered with advancing age. With respect to the so-called indolent lymphomas, a decreasing complete remission rate and overall survival rate (5-year) was observed for patients older than 70 years, whereas patients with DLBL fared worse when older than 65 years and 60 years, respectively. The age-adjusted IPI score was discriminative for prognosis. However, even with an IPI score nil, the age group older than 75 years fared significantly worse (P < 0.009), but less so with the relative survival model. The relative survival at 5 years was 60%, 53%, 48%, 35%, and 32% for the 5 respective age groups. CONCLUSIONS Patients with indolent lymphomas become elderly when they are older than 70 years, but when aggressive lymphoma is concerned this occurs when patients are older than 65 years. For patients with an IPI score nil, age older than 75 years is the dominant prognostic factor. The negative influence of concomitant disease on overall survival, although continuously increasing in older age groups, seems to diminish for patients older than 75 years when compared with the general Dutch population. Cancer 2000;89:2667–76. © 2000 American Cancer Society.

Langerhans cells (LC) represent the dendritic cell (DC) lineage in the epidermis. They capture and process antigens in the skin and subsequently migrate to the draining lymph nodes to activate naive T cells. Efficient uptake and processing of protein antigens by LC would, therefore, seem a prerequisite. We have now compared the capacity of human epidermal LC, blood-derived DC and peripheral blood mononuclear cells to endocytose and present (mannosylated) antigens to antigen-specific T cells. Moreover, we have determined the expression of mannose receptors, and the composition of the intracellular endosomal/lysosomal MHC class II-positive compartment. The results indicate that LC have poor endocytic capacity and do not exploit mannose receptor-mediated endocytosis pathways. Furthermore, the composition of the class II compartment in LC is distinct from that in other antigen-presenting cells and is characterized by the presence of relatively low levels of lysosomal markers. These results underscore the unique properties of LC and indicate that LC are relatively inefficient in antigen uptake, processing and presentation. This may serve to avoid hyper-responsiveness to harmless protein antigens that are likely to be frequently encountered in the skin due to (mechanical) skin damage.

We describe a patient with a primary diffuse large B-cell lymphoma of the central nervous system who developed a localized testicular relapse after 8 years. Both tumours lacked HLA-DR expression, the relapse additionally lost HLA class I expression. Immunoglobulin heavy chain gene rearrangements were identical in both lymphomas with extensive and ongoing somatic hypermutations resulting in extensive idiotype modulation. We hypothesize that these immune sanctuaries initially provided a safe haven for the tumour cells. When the environment becomes more permissive for an anti-tumour response, the continuous idiotype modulation and progressive loss of HLA expression on the tumour cells facilitates further immune escape.

BACKGROUND Primary extranodal lymphomas (EN-NHLs) are a heterogeneous category of tumors that are considered to be different from primary nodal non-Hodgkin's lymphomas (N-NHLs). To what extent these differences have clinical implications is currently not very clear, because knowledge of EN-NHL as a separate group is limited. METHODS Using data from the Comprehensive Cancer Centre West (CCCW) population-based NHL registry in the Netherlands, N-NHL and EN-NHL patients were compared to determine differences in characteristics at diagnosis, responses to treatment, patterns of failure, and survival. RESULTS At presentation, EN-NHL patients had poorer performance scores and more often bulky tumors compared with N-NHL patients, resulting in poorer responses to treatment (complete response rates were 72% and 84%, respectively; P = 0.04) and inferior 5-year overall survival (49% and 63%, respectively; P = 0.003). Among EN-NHL patients, considerable variations in response, survival, and relapse rates were observed, with gastric NHL patients having the best and central nervous system NHL patients having the worst prognosis (66% and 7% 5-year overall survival, respectively). Relapse rates for N-NHL and EN-NHL patients did not differ (39% and 36% 5-year relapse rates, respectively), whereas among EN-NHL patients considerable differences in relapse rates were noted. Relapses among N-NHL patients were mainly found in nodal sites, whereas recurrent disease in EN-NHL patients was mainly found in extranodal sites. CONCLUSIONS In this population-based study, Stage I EN-NHL patients as a group had a poorer prognosis than N-NHL patients. However, among EN-NHL patients, considerable differences in response, relapse risk, and survival were observed. The failure analysis conducted in this study suggests that patterns of dissemination for N-NHL and EN-NHL are different. Cancer 1998;83:1612-1619. © 1998 American Cancer Society.

Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cμ-Cδ genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cγ gene or pseudogene in the 3′ cluster. © 1998 by The American Society of Hematology.

We have identified a novel gene, EMS1, that is consistently amplified and overexpressed in human carcinomas with an amplification of the chromosome 11q13 region. Comparisons of the EMS1 sequences with those present in the GenBank databases revealed a high identity with chicken cortactin. Southern and western blot analyses confirm the high sequence conservation during evolution. An antiserum specific for human cortactin, showed in gene transfer experiments that both human p80 and p85 isoforms are encoded by the EMS1 cDNA. Further comparisons demonstrated an high sequence and structural homology with HS1 that is implicated in signal transduction in lymphoid cells only. Expression of EMS1/cortactin mRNA was restricted to tumor cell lines derived from non-lymphoid origin. Cortactin contains (i) a filamentous actin binding tandem repeat domain, (ii) a proline-rich SH3-binding and (iii) a SH3 domain that is common in proteins involved in signal transduction. Our data suggest that human EMS1/cortactin has a function in signal transmission between cell-matrix contact sites and the cytoskeleton and, as such, its overexpression due to 11q13 amplification might effect adhesive properties of human carcinomas.

Recently, a new player in the cytokine network has been described that is produced by monocytes and can be detected in the rheumatoid synovium: interleukin-15 (IL-15). Since this cytokine may play a role in the accumulation and activation of T-cells, B-cells, and natural killer (NK) cells characteristic of synovial tissue (ST) from patients with rheumatoid arthritis (RA), the expression of IL-15 was studied in ST from RA patients in comparison with ST from patients with reactive arthritis (ReA) and osteoarthritis (OA) and the phenotype of IL-15-positive cells was determined. IL-15 expression was investigated by immunohistochemical analysis of ST from ten patients with RA, ten patients with Yersinia enterocolitica-induced ReA, and nine patients with OA. The immunohistological findings were quantified and the results obtained in the different patient groups were compared. To determine the phenotype of IL-15-expressing cells, double-labelling immunofluorescence was performed. The expression of IL-15 was significantly higher in ST from patients with RA than in ST from patients with ReA or OA. In double-label experiments, co-expression was observed with markers for macrophages, T-cells, and NK cells. The composition of the cellular infiltrate in the synovium of patients with RA might be partly explained by the specific increase in expression of IL-15 in rheumatoid ST. It can be speculated that IL-15 production by inflammatory cells other than macrophages may occur in the rheumatoid synovium.

BACKGROUND Non-Hodgkin's lymphoma (NHL) originating in mucosa-associated lymphoid tissue (MALT) is supposed to have different clinical behavior from lymph node NHL. To test this hypothesis, the authors compared data of gastric NHL patients with lymph node NHL patients in a population-based registry for differences in clinical presentation and prognosis. METHODS Data from 1981-1989 on patients with primary gastric NHL (n = 109) and patients with primary lymph node NHL (n = 658) were retrieved from a Dutch population-based NHL registry. Patients were compared for stage distribution, involved sites, and survival. The prognostic value of grading lymphomas according to the malignancy grades of the Working Formulation for Clinical Usage was compared with the value of grading MALT NHLs as either low grade or high grade malignancies. RESULTS Patients with gastric NHL presented more often with localized disease. Stage IV patients had a higher rate of dissemination to other non-lymph node sites but less frequent localization in the bone marrow. The restricted pattern of dissemination was reflected in a significantly lower recurrence rate for gastric NHL. Gastric NHL patients had significantly better disease free survival than lymph node NHL patients (80% and 44% at 5 years, respectively; P < 0.001). In contrast, overall survival did not significantly differ between the two groups, and it appeared to depend on disease stage. Grading MALT lymphoma as either low grade (26%) or high grade (70%) malignancies did not provide better prognostic information than grading according to the Working Formulation for Clinical Usage (low 8%, intermediate 75%, high 9%). CONCLUSIONS Primary gastric NHL shows a restricted dissemination pattern, which gives support to the MALT lymphoma concept. Although this might explain the superior disease free survival observed for gastric NHL patients, it does not translate into better overall survival for these patients. Cancer 1997; 79:390-7. © 1997 American Cancer Society.

The increasing use of immunophenotypic and molecular techniques on lymphoid tissue samples without obvious involvement by malignant lymphoma has resulted in the increased detection of "early" lymphoid proliferations, which show some, but not all the criteria necessary for a diagnosis of malignant lymphoma. In most instances, these are incidental findings in asymptomatic individuals, and their biological behaviour is uncertain. In order to better characterize these premalignant conditions and to establish diagnostic criteria, a joint workshop of the European Association for Haematopathology and the Society of Hematopathology was held in Uppsala, Sweden, in September 2010. The panel reviewed and discussed more than 130 submitted cases and reached consensus diagnoses. Cases representing the nodal equivalent of monoclonal B-cell lymphocytosis (MBL) were discussed, as well as the "in situ" counterparts of follicular lymphoma (FL) and mantle cell lymphoma (MCL), topics that also stimulated discussions concerning the best terminology for these lesions. The workshop also addressed the borderland between reactive hyperplasia, and clonal proliferations such as pediatric marginal zone lymphoma and pediatric FL, which may have very limited capacity for progression. Virus-driven lymphoproliferations in the grey zone between reactive lesions and manifest malignant lymphoma were covered. Finally, early manifestations of T-cell lymphoma, both nodal and extranodal, and their mimics were addressed. This workshop report summarizes the most important conclusions concerning diagnostic features, as well as proposals for terminology and classification of early lymphoproliferations and tries to give some practical guidelines for diagnosis and reporting.

Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). This study was undertaken to examine the distribution of defined B cell subsets, including effector B (Beff) cells and regulatory B (Breg) cells, in patients with GCA and patients with PMR before and after corticosteroid treatment.Circulating B cells were analyzed in 34 newly diagnosed, untreated patients with GCA or PMR, and in 44 followup samples from patients with GCA or PMR who received corticosteroids for 2 weeks or 3 months. For comparison, 40 age-matched healthy controls and 11 rheumatoid arthritis (RA) patients were included. Serum BAFF levels were determined, and temporal arteries were studied by immunohistochemistry.Patients newly diagnosed as having GCA or PMR, but not patients with RA, had decreased numbers of circulating B cells compared to healthy controls. B cell numbers recovered rapidly in treated patients with GCA and PMR in remission. This recovery was not achieved by compensatory hyperproliferation or enhanced bone marrow production. B cell numbers inversely correlated with erythrocyte sedimentation rates, C-reactive protein levels, and serum BAFF levels. Tumor necrosis factor α-positive Beff cells, but not interleukin-10 (IL-10)-positive Breg cells, were decreased in patients newly diagnosed as having GCA or PMR. Following treatment, circulating numbers of Beff cells normalized. The returning Beff cells demonstrated an enhanced capacity to produce IL-6. Few B cells were found in temporal artery biopsy specimens from GCA patients.We show for the first time that the distribution of B cells is highly disturbed in GCA and PMR and that B cells likely contribute to the enhanced IL-6 response in both diseases.

Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.

Contains fulltext : 178096.pdf (Publisher’s version ) (Open Access) Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis. 01 oktober 2013

The objective of this paper is to define the expression of adhesion molecules in synovial tissue (ST) from patients with rheumatoid arthritis (RA) with respect to disease duration. Antibodies against adhesion molecules were used for immunohistochemistry to examine ST sections from 11 patients with early RA (1 year), 14 patients with long-standing RA (5 years), and 15 patients with osteoarthritis (OA). Increased cellular infiltration and increased expression of E-selectin, ICAM-1, VCAM-1, PECAM-1, VLA-4, and Mac-1 were found in ST from patients with RA compared to ST from patients with OA. The immunohistological findings were similar for the different stages of RA. The upregulation of adhesion molecules in ST of patients with RA of1 year's duration suggests the activation of chronic inflammatory processes in these patients. Therefore, the mechanisms by which therapies directed toward these adhesion molecules exert their effects are likely to be similar for patients with so-called early RA and patients with long-standing RA.

Background The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In app. 10% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CARD11. Recent evidence suggests that autoantigen recognition by the BCR sustains the viability of ABC DLBCL in vitro (Young, PNAS 2015), although the functional relationship to CARD11 mutations remains unresolved. Antigen-independent, constitutively active BCR signaling is a fundamental oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We therefore explored the hypothesis that autonomous BCR signaling akin to CLL could be operative in ABC DLBCL and would represent an alternative but functionally complementary oncogenic mechanism to activating CARD11 mutations in ABC DLBCL lymphomagenesis. Methods BCR transcripts were identified from archived fresh-frozen biopsies of 14 histologically confirmed, CD10-negative, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased isolation of full-length BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR were identified by single molecule sequencing on the PacBio platform. All biopsies were screened for mutations in CARD11 and CD79 by Sanger sequencing. To test for autonomous BCR signaling activity, murine triple KO (TKO) cells were transduced with functional DLBCL BCR (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon restoring functionality of the BCR signalling cascade with tamoxifen as calcium flux by flow cytometry and robust cellular proliferation. Results Re-examination by immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6 was possible in 12 cases and identified three cases as GCB-type DLBCL. Nine cases were classified as non-GCB DLBCL, including one primary CNS DLBCL and three leg-type primary cutaneous DLBCL (LT DLBCL). One non-GCB DLBCL was found to carry a potentially activating CARD11 M212K mutation (Table). BCR of the three GCB DLBCL served as controls and lacked an autonomous BCR signal. In contrast, TKO cells transduced with five of the seven remaining DLBCL BCR (71%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. In accordance with our hypotheses, the CARD11-mutated non-GCB DLBCL case lacked autonomous BCR signaling. This case and the second, non-GCB DLBCL case lacking autonomous BR activity that carried wild-type CARD11 both expressed a BCR with the IGHV4-34 allele. Unexpectedly, the three LT DLBCL also did not exhibit autonomous BCR signaling. Conclusion Our data demonstrate autonomous BCR activity in a dominant fraction of non-GCB DLBCL. In striking similarity to CLL, these results point to an oncogenic role of a structurally normal BCR in non-GCB DLBCL that does not require binding of external autoantigens. Consistent with our hypotheses, antigen-independent signaling of a structurally normal BCR signaling cascade is a compelling candidate alternative mechanism to activating mutations in signal-transducing components of the BCR pathway. Both mechanisms may therefore induce the characteristic gene expression signature of ABC DLBCL. In addition, this finding readily explains the higher likelihood of ABC DLBCL with wild-type CD79 and CARD11 to respond to BCR signaling inhibition by BTK inhibitors (Wilson, Nat Med 2015). In contrast, LT DLBCL, which otherwise resembles ABC DLBCL, does not exhibit autonomous BCR signaling, suggesting yet another oncogenic mechanism that may explain its dismal prognosis. Finally, the non-GCB DLBCL lacking both autonomous BCR signaling and a potentially activating CARD11 mutation expressed the inherently autoreactive IGHV4-34 allele, opening the possibility that strong autoantigen stimulation might substitute for autonomous BCR signaling activity, thereby offering an alternative explanation for the BCR signaling pathway addiction in IGHV4-34-expressing cases. Table. Table. Disclosures Vermeer: Innate Pharma: Other: Investigator in a clinical trial.

Background Primary mediastinal large B-cell lymphoma (PM LBCL) is putatively derived from thymic B cells. PM LBCL is clinically and morphologically distinct from diffuse large B-cell lymphoma (DLBCL), but shares certain phenotypic characteristics with nodular sclerosing classical Hodgkin's lymphoma. The expression and function of an intact B-cell receptor (BCR) in PM LBCL is controversial. In addition, the pathological mechanisms that drive lymphomagenesis in PM DLBCL are largely unknown. Here, we investigate the expression of functional BCR in PM LBCL and explore the hypothesis that autonomous signaling of the BCR as observed in CLL (Dühren-von Minden, Nature 2012) plays a key role in PM DLBCL pathogenesis. Methods The PM LBCL cell lines Farage, MedB-1, Karpas 1106P, and U2940 were tested in vitro by comparing calcium flux with and without inhibition of BCR signaling by blockade of Syk with the tyrosine kinase inhibitor R406. BCR transcripts were identified from archived fresh-frozen biopsies from two histologically confirmed PM LBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. To test for autonomous BCR signaling, murine triple KO (TKO) cells were transduced with functional BCR of PM LBCL (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon induction with tamoxifen as calcium flux by flow cytometry or as robust cellular proliferation. Results All four PM-LBCL lines had higher calcium flux in the absence of R406, suggesting autonomous BCR signaling activity without antigenic stimulation or artificial BCR crosslinking. Functional BCR transcripts could be readily identified in both primary PM LBCL biopsies by ARTISAN PCR. In accordance with our hypothesis, TKO cells transduced with the BCR of both primary PM LBCL cases showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. To test whether this autonomous BCR signal would represent a druggable therapeutic target, the PM DLBCL cell lines were cultured in the presence of increasing doses of the second generation, highly BTK-specific kinase inhibitor acalabrutinib. The ABC DLBCL cell lines OCI-Ly10, TMD8, and U2932, and the GCB cell lines Karpas 422, OCI-Ly7, and OCI-Ly19 were tested in parallel as controls. Cell viability decreased in all cell lines with increasing concentrations of acalabrutinib (Figure). Consistent with their known dependency on active BCR signalling (Young, PNAS 2015; Koning, ASH 2016), ABC DLBCL cell lines were very sensitive to acalabrutinib. As suggested by the experimental evidence for autonomous BCR signalling, PM LBCL cell lines also responded to acalabrutinib, albeit to a lesser degree than ABC DLBCL. Growth inhibition of GCB DLBCL cell lines generally required excessively high doses of acalabrutinib, consistent with the notion that this DLBCL subtype depends on tonic BCR signaling only. Conclusion Consistent with our hypothesis, our data demonstrate expression of an intact BCR with autonomous signaling activity in all investigated cases of PM LBCL. These results point to an oncogenic role of structurally normal BCR in PM LBCL akin to CLL. Similarly to CLL and ABC DLBCL, PM DLBCL cell lines respond to BTK inhibitors, albeit less strongly than the ABC DLBCL cell lines tested. The results identify BCR signaling as a novel therapeutic target in PM LBCL and suggest the initiation of clinical trials with BTK inhibitors in PM LBCL. Figure Figure. Disclosures No relevant conflicts of interest to declare.

The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In up to 30% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CD79A, CD79B, or CARD11. Antigen-independent, constitutively active (BCR) signaling is a general oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We hypothesized that autonomous BCR signaling akin to CLL may operate in ABC DLBCL and could explain the characteristic ABC gene expression signature. BCR transcripts were identified from fresh-frozen biopsies from 12 histologically confirmed, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching. Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. Triple KO (TKO) cells were transduced with functional DLBCL BCR. TKO cells are arrested at the pre-B-cell stage due to lack of the rag2 and lambda5 genes. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65. When transduced with expression constructs encoding a functional BCR, autonomous or antigen-induced BCR signalling can be measured as calcium flux upon induction with tamoxifen. TKO cells transduced with seven of the twelve DLBCL BCR (58%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. By histomorphology and immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6, these cases were classified as non-GCB-type DLBCL, including one primary CNS DLBCL and two primary mediastinal (PM-)DLBCL. In accordance with our hypothesis, BCR from three DLBCL classified as GCB lacked an autonomous BCR signal. The remaining two negative cases were non-GCB-type and are currently being analyzed for activating CD79/CARD11 mutations. To further explore the detection of autonomous BCR signaling in DLBCL, PM-DLBCL and ABC DLBCL cell lines were tested in vitro by comparing calcium flux with and without blockade of Syk signaling by the tyrosine kinase inhibitor R406. All four PM-DLBCL and two of five ABC DLBCL cell lines had higher calcium flux in the absence of R406, indicating autonomous signaling activity without antigenic stimulation or artificial BCR crosslinking. In summary, we demonstrate autonomous BCR activity in a substantial fraction (72%) of non-GCB DLBCL (including PM-DLBCL). These results point to an oncogenic role of structurally normal BCR in non-GCB DLBCL akin to CLL. Autonomous BCR signaling is a candidate alternative mechanism to the previously described activating mutations of components of the BCR signaling cascade to induce the characteristic gene expression signature of ABC DLBCL. Formal delineation of autonomous BCR signalling to activating mutations in the BCR signalling cascade is ongoing. Citation Format: Marvyn T. Koning, Rudolf Übelhart, Arjen H.G. Cleven, Willem H. Zoutman, Sander A.J. van der Zeeuw, Philip Kluin, Marieke Griffioen, Hans G. Drexler, Cornelis P. Tensen, Maarten H. Vermeer, Rein Willemze, Szymon M. Kielbasa, Marcelo A. Navarrete, Hassan Jumaa, Hendrik Veelken. Autonomous, antigen-independent B-cell receptor signalling as a novel pathogenetic mechanism in non-GCB DLBCL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-012.

Objective. To define the expression and the phenotype of granzyme (Gran) A and B positive cytotoxic cells in synovial tissue (ST) from patients with rheumatoid arthritis (RA) with respect to disease duration and activity. Methods. Using antibodies against GranA and GranB, which serve as markers of activated natural killer (NK) cells and cytotoxic T lymphocytes, ST sections from 10 patients with early RA, 10 patients with longstanding RA, and 10 patients with osteoarthritis were examined. The phenotype of Gran+ cells was determined with double-labeling techniques. Results. Gran+ cells, the majority of which were NK cells, were found in ST from patients in all groups. Several of these cells did not express the surface markers CD16, CD56, and CD57. The highest ST expression of GranB was found in patients with early RA. In RA patients, there was a positive correlation of GranB expression with serum levels of acute-phase reactants, but not with histologic scores for inflammation. Conclusion. Gran+ cells are mainly NK cells, a substantial proportion of which do not express conventional NK cell surface markers. GranB expression is specifically increased in the synovial tissues of patients with RA of short duration.

In order to identify acute myeloid leukemia (AML) CD34(+)-specific gene expression profiles, mononuclear cells from AML patients (n = 46) were sorted into CD34(+) and CD34(-) subfractions, and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34(+) and CD34(-) gene expression was compared with a large group of normal CD34(+) bone marrow (BM) cells (n = 31). Unsupervised hierarchical clustering analysis showed that CD34(+) AML samples belonged to a distinct cluster compared with normal BM and that in 61% of the cases the AML CD34(+) transcriptome did not cluster together with the paired CD34(-) transcriptome. The top 50 of AML CD34(+)-specific genes was selected by comparing the AML CD34(+) transcriptome with the AML CD34(-) and CD34(+) normal BM transcriptomes. Interestingly, for three of these genes, that is, ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2), a high transcript level was associated with a significant poorer overall survival (OS) in two independent cohorts (n = 163 and n = 218) of normal karyotype AML. Importantly, the prognostic value of the continuous transcript levels of ANKRD28 (OS hazard ratio (HR): 1.32, P = 0.008), GNA15 (OS HR: 1.22, P = 0.033) and UGP2 (OS HR: 1.86, P = 0.009) was shown to be independent from the well-known risk factors FLT3-ITD, NPM1c(+) and CEBPA mutation status. Leukemia (2011) 25, 1825-1833; doi:10.1038/leu.2011.172; published online 15 July 2011

Rationale: A discordant relationship between bone marrow cellularity and peripheral blood findings is regularly noticed in patients with aplastic anemia (AA). Therefore, the feasibility of 3-F-18 fluoro-3-deoxy-L-thymidine (F-18 FLT PET was tested as a noninvasive tool to visualize the total distribution of the hematopoietic bone marrow compartment in AA at presentation or after treatment.Methods: In vivo scanning was performed with F-18 FLT PET in AA patients (n = 17), including patients upfront (n = 11) and following treatment (n = 6), in addition to peripheral blood cell counts and a bone marrow biopsy.Results: A striking abnormal F-18 FLT scan was observed in all patients upfront treatment, in particular a reduced uptake of the pelvis was shown, the area that is biopsied for the bone marrow biopsy. Following treatment, the number of solitary lesions with increased proliferative activity outside the pelvis was noticed in patients with partial response, whereas patients with a complete remission showed a homogenous uptake throughout the skeleton.Conclusion: This pilot study demonstrates that F-18 FLT scan provides a highly distinctive overview of the bone marrow compartment in AA that might be helpful for making a proper diagnosis and monitoring treatment response of AA patients.