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Key Points Diagnosis of TCL often relies on PCR-based TCR clonality testing, which displays low specificity and requires high-quality DNA.Targeted NGS and T-cell clonality assessment are complementary techniques that should be applied in hard front-line diagnosis., Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques’ performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4(+) T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient–derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK–transformed CD4(+) T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients’ anaplastic large cell lymphomas. Integration of “Omic” data revealed that NPM-ALK–transformed CD4(+) T lymphocytes and primary NPM-ALK(+) ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK(+) lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK(+) cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30(+) peripheral CD4(+) T cells, in keeping with a thymic progenitor-like pattern.

AimsPrimary mediastinal large B-cell lymphoma (PMBL) diagnosis can be challenging on needle biopsies. Robust techniques are needed to ensure diagnosis of this lymphoma which is highly sensitive to recently developed therapy protocols.MethodsIn this study, we sought to determine precise PMBL phenotype, compared with diffuse large B-cell lymphoma not otherwise specified, by combining immunohistochemistry with anti-MAL antibody and RNA in situ hybridisation (RNAscope) with specific MAL probes.ResultsThe overall MAL positivity level reached 93% (14/15) of cases of PMBL. Among the 15 cases enrolled in the study, 11 were undoubtedly positive for MAL immunostaining whereas 13 were positive by RNA in situ hybridisation. Interestingly, one case that was negative by in situ hybridisation turned out to be positive by immunohistochemistry.ConclusionsTaken together, our results demonstrate that in situ detection of both MAL transcripts and protein are complementary and increase the sensitivity and specificity of PMBL diagnosis.

T-cell lymphomas (TCL) represent a very heterogeneous group of lymphoid tumors which are clearly distinct from B-cell neoplasms [...]

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition–inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. Significance: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ. See related commentary by Carvajal-Maldonado and Wood, p. 1441

International audience; The most frequent genetic alteration in acute myeloid leukemia (AML) is the mutation of nucleophosmin 1 (NPM1). Yet, its downstream oncogenic routes are not fully understood. Here, we report the identification of one long noncoding RNA (lncRNA) overexpressed in NPM1-mutated AML patients (named LONA) whose intracellular localization inversely reflects that of NPM1. While NPM1 is nuclear and LONA cytoplasmic in wild-type NPM1 AML cells, LONA becomes nuclear as mutant NPM1 moves toward the cytoplasm. Gain or loss of function combined with a genome-wide RNA-seq search identified a set of LONA mRNA targets encoding proteins involved in myeloid cell differentiation (including THSB1, MAFB, and ASB2) and interaction with its microenvironment. Consistently, LONA overexpression in mutant NPM1 established cell lines and primary AML cells exerts an anti-myeloid differentiation effect, whilst it exerts an opposite promyeloid differentiation effect in a wild type NPM1 setting. In vivo, LONA overexpression acts as an oncogenic lncRNA reducing the survival of mice transplanted with AML cells and rendering AML tumors more resistant to AraC chemotherapy. These data indicate that mutation-dependent nuclear export of NPM1 leads to nuclear retention and consequent oncogenic functions of the overexpressed lncRNA LONA, thus uncovering a novel NPM1 mutation-dependent pathway in AML pathogenesis.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. The update of the 4th edition of the 2017 WHO classification provides new concepts in the classification of DLBCL with the definition of 2 new categories : High-grade B cell lymphoma (HGBL) with MYC, BCL2 and/or BCL6 rearrangements so-called double-hit (DH) or triple-hit (TH) lymphomas, and HGBL, not otherwise specified (NOS) without MYC and BCL2 or BCL6 translocations [1] . Patients with MYC/BCL2 DHL, MYC/BCL6 DHL or MYC/BCL2/BCL6 THL will usually have an aggressive clinical course. We report here genomic characterization of 20 adult patients diagnosed with DHL harboring MYC and BCL2 (n = 15) or MYC and BCL6 (n = 2) rearrangements and 3 THL. To identify molecular variants, we performed targeted next generation sequencing (NGS) of FFPE samples of DHL/THL for mutations on 43 genes known to be important for lymphomagenesis. In all FFPE DHL/THL cases, our NGS Lymphopanel identified 438 alterations on 40 genes. Of these, 197/438 (45%) alterations localized on 33 genes are non-synonymous and predict amino-acid substitutions or truncation of the proteins. All samples harbor non-synonymous somatic alterations (5 to 18). The most frequently mutated genes are CREBBP (16/20 cases) followed by BCL2 (12/20), KMT2D (12/20), MYC (9/20), EZH2 (8/20), IGLL5 (8/20), FOXO1 (6/20) and SOCS1 (6/20). We also assessed the impact of AID involvement and found that IGLL5, SOCS1, MYC and BCL2 have the highest AID mutation frequency (40%, 38%, 30% and 27%, respectively) whereas KMT2D, FOXO1 and EZH2 show no AID induced mutations. Furthermore, DHL/THL mutations are mainly involved in apoptosis/cell-cycle (35% of total variants) and epigenetic regulation pathways (32% of total variants), known to be associated with a poor prognosis in lymphoma patients. In conclusion, our study describes for the first time the mutational landscape of DHL/THL, a poorly studied subtype of aggressive B cell lymphoma [2] .

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloblastic leukaemia. We previously described a recurrent t(X;6)(p11;q23) translocation generating an MYB-GATA1 fusion gene in male infants with ABL. To better understand its role, the chimeric MYB-GATA1 transcription factor was expressed in CD34-positive haematopoietic progenitors, which were transplanted into immunodeficient mice. Cells expressing MYB-GATA1 showed increased expression of markers of immaturity (CD34), of granulocytic lineage (CD33 and CD117), and of basophilic differentiation (CD203c and FcϵRI). UT-7 cells also showed basophilic differentiation after MYB-GATA1 transfection. A transcriptomic study identified nine genes deregulated by both MYB-GATA1 and basophilic differentiation. Induction of three of these genes (CCL23, IL1RL1, and NTRK1) was confirmed in MYB-GATA1-expressing CD34-positive cells by reverse transcription quantitative polymerase chain reaction. Interleukin (IL)-33 and nerve growth factor (NGF), the ligands of IL-1 receptor-like 1 (IL1RL1) and neurotrophic receptor tyrosine kinase 1 (NTRK1), respectively, enhanced the basophilic differentiation of MYB-GATA1-expressing UT-7 cells, thus demonstrating the importance of this pathway in the basophilic differentiation of leukaemic cells and CD34-positive primary cells. Finally, gene reporter assays confirmed that MYB and MYB-GATA1 directly activated NTRK1 and IL1RL1 transcription, leading to basophilic skewing of the blasts. MYB-GATA1 is more efficient than MYB, because of better stability. Our results highlight the role of IL-33 and NGF receptors in the basophilic differentiation of normal and leukaemic cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity. Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1. Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer. Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration. Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies. Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy., There are many patients who do not respond to immune checkpoint inhibitor (ICI) immunotherapy. Here, the authors show a significant negative correlation between sphingosine kinase-1 (SK1) expression and survival for ICI-treated melanoma patients, and further show that targeting SK1 improves response to ICI in mouse cancer models.

Background Immunotherapy is currently under investigation in B3 Thymoma (TB3) and Thymic Carcinoma (TC). PD-L1 expression has been evaluated on a limited number of patients with selected antibodies. We aimed to analyze cohort of TB3 and TC with a panel of antibodies to assess the prevalence of PD-L1 expression, its prognostic value and to set up a reproducible test. Methods We retrospectively studied 103 patients samples of FFPE histologically confirmed TB3 (n = 53) and TC (n = 50) by expert pathologists within the RYTHMIC national network. We compared PD-L1, PD1, CD8 and PD-L2 expression and performed correlation with tumor types and patients outcomes. Four PD-L1 antibodies were tested, three of them validated as companion tests in lung cancer, one tested on two automates on whole section of tumors. We evaluated the percentage and intensity of both epithelial and immune stained cells. Results TB3 epithelial cells had a higher and more diffuse expression of PD-L1 than TC regardless the antibodies tested (p Conclusion Frequent PD-L1 expression, particularly in TB3, paves the way for immunotherapy in TET (Thymic Epithelial Tumor). Otherwise, we have set up three reproducible LDT (laboratory-developed test) for four PD-L1 antibodies.

We previously reported a new form of light chain deposition disease (LCDD) presenting as diffuse cystic lung disorder that differs from the usual systemic form with respect to patient age, the male/female ratio, the involved organs, and the hematologic characteristics. We also demonstrated that the light chains were produced by an intrapulmonary B-cell clone and that this clone shared a stereotyped antigen receptor IGHV4-34/IGKV1. However, we only analyzed 3 patients. We conducted a retrospective study including lung tissue samples from 24 patients with pulmonary LCDD (pLCDD) matched with samples from 13 patients with pulmonary κ light chain amyloidosis (pAL amyloidosis) used as controls. Mass spectrometry–based proteomics identified immunoglobulin κ peptides as the main protein component of the tissue deposits in all patients. Interestingly, in pLCDD, IGKV1 was the most common κ family detected (86.4%), and IGKV1-8 was overrepresented compared with pAL amyloidosis (75% vs 11.1%, P = .0033). Furthermore, IGKV1-8 was predominantly associated with a diffuse cystic pattern (94%) in pLCDD. In conclusion, the high frequency of IGKV1-8 usage in cystic pLCDD constitutes an additional feature arguing for a specific entity distinct from the systemic form that preferentially uses IGKV4-1.

International audience; Anaplastic large-cell lymphoma, a T-cell neoplasm, is primarily a pediatric disease. Seventy-five percent of pediatric anaplastic large-cell lymphoma cases harbor the chromosomal translocation t(2;5)(p23;q35) leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. NPM-ALK consists of an N-terminal nucleophosmin (NPM) domain fused to an anaplastic lymphoma kinase (ALK) cytoplasmic domain. Pediatric NPM-ALK+ anaplastic large-cell lymphoma is often a disseminated disease and young patients are prone to chemoresistance or relapse shortly after chemotherapeutic treatment. Furthermore, there is no gold standard protocol for the treatment of relapses. To the best of our knowledge, this is the first study on the potential role of the microRNA, miR-497, in NPM-ALK+ anaplastic large-cell lymphoma tumorigenesis. Our results show that miR-497 expression is repressed in NPM-ALK+ cell lines and patient samples through the hypermethylation of its promoter and the activity of NPM-ALK is responsible for this epigenetic repression. We demonstrate that overexpression of miR-497 in human NPM-ALK+ anaplastic large-cell lymphoma cells inhibits cellular growth and causes cell cycle arrest by targeting CDK6, E2F3 and CCNE1, the three regulators of the G1 phase of the cell cycle. Interestingly, we show that a scoring system based on CDK6, E2F3 and CCNE1 expression could help to identify relapsing pediatric patients. In addition, we demonstrate the sensitivity of NPM-ALK+ cells to CDK4/6 inhibition using for the first time a selective inhibitor, palbociclib. Together, our findings suggest that CDK6 could be a therapeutic target for the development of future treatments for NPM-ALK+ anaplastic large-cell lymphoma.

International audience; Amyloidoses are rare life-threatening diseases caused by protein misfolding of normally soluble proteins. The fatal outcome is predominantly due to renal failure and/or cardiac dysfunction. Because amyloid fibrils formed by all amyloidogenic proteins share structural similarity, amyloidoses may be studied in transgenic models expressing any amyloidogenic protein. Here we generated transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII, a major protein of high density lipoprotein. According to amyloid nomenclature this variant was termed STOP78SERApoAII. STOP78SER-APOA2 expression at the physiological level spontaneously induced systemic amyloidosis in all mice with full-length mature STOP78SERApoAII identified as the amyloidogenic protein. Amyloid deposits stained with Congo red, were extracellular, and consisted of fibrils of approximately 10 nm diameter. Renal glomerular amyloidosis was a major feature with onset of renal insufficiency occurring in mice older than six months of age. The liver, heart and spleen were also greatly affected. Expression of STOP78SERAPOA2 in liver and intestine in mice of the K line but not in other amyloid-laden organs showed they present systemic amyloidosis. The amyloid burden was a function of STOP78SER-APOA2expression and age of the mice with amyloid deposition starting in two-month old highexpressing mice that died from six months onwards. Because STOP78SER-ApoAII conserved adequate lipid binding capacity as shown by high STOP78SER-ApoAII amounts in high density lipoprotein of young mice, its decrease in circulation with age suggests preferential deposition into preformed fibrils. Thus, our mouse model faithfully reproduces early-onset hereditary systemic amyloidosis and is ideally suited to devise and test novel therapie

International audience; PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in BALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human BALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in BALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN-expressing mice efficiently developed BALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN-regulated gene candidates that establish the molecular bases of the pre-leukemic state to drive BALL initiation. Hence, our study provides a new in vivo model of human BALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

Therapeutic blockade of PD-1/PD-L1 shows promising results in Hodgkin&rsquo, s lymphoma (HL) and in some diffuse large B-cell lymphoma (DLBCL) patients, but biomarkers predicting such responses are still lacking. To this end, we recently developed a transcriptional scoring of immune escape (IE) in cancer biopsies. Using this method in DLBCL, we identified four stages of IE correlated with overall survival, but whether Hodgkin&rsquo, s lymphomas (HL) also display this partition was unknown. Thus, we explored the transcriptomic profiles of ~1000 HL and DLBCL using a comparative meta-analysis of their bulk microarrays. Relative to DLBCL, the HL co-clustered at the advanced stage of immune escape, displaying significant enrichment of both IE and T-cell activation genes. Analyses via transcriptome deconvolution and immunohistochemistry showed more CD3+ and CD4+ tumor-infiltrating lymphocytes (TILs) in HL than DLBCL. Both HL and non-GCB DLBCL shared a high abundance of infiltrating CD8+ T-cells, but HL had less CD68+CD163+ macrophages. The same cellular distribution of PD-1 and TIM-3 was observed in HL and DLBCL, though HL had more PD-L1 tumor cells and LAG-3 ME cells. This study illuminates the advanced stage of immune activation and escape in HL, consistent with the response to checkpoint blockade therapies for this type of lymphoma.

International audience; Langerhans cell histiocytosis (LCH) has a mostly favorable outcome, whereas Langerhans cell sarcoma (LCS) is an aggressive tumor. It is still unclear whether any specific molecular alterations could underlie the aggressive behavior of Lan-gerhans cell proliferations. We used targeted next-generation sequencing and array-comparative genomic hybridization to profile 22 LCH samples from different patients together with 3 LCS samples corresponding to different relapses from the same patient. The third LCS relapse was a composite tumor including both B-cell chronic lymphocytic leukemia and LCS components. The 22 LCH samples were mostly of bone origin and showed classic histophenotypical features. Array-comparative genomic hybridization showed in all 3 LCS samples a similar homozygous somatic loss affecting the CDKN2A/B locus, whereas the 17 informative LCH samples did not show any detectable abnormality. In the 3 LCS samples, targeted next-generation sequencing of 495 cancer genes detected common mutations in KMT2D/MLL2 and in both MAP2K1 and NRAS genes, whereas BRAF was not mutated. A NOTCH1 mutation was acquired in 2 LCS samples. The composite LCS/B-cell chronic lymphocytic leukemia tumor showed the same genetic profile in its 2 components. LCH samples showed mutually exclusive mutations of BRAF (8/20) and MAP2K1 (4/19), but no mutation of KMT2D, NRAS nor NOTCH1. These results suggest that CDKN2A/B deletion and/or simultaneous mutations of MAP2K1 and NRAS may underlie the aggressive behavior of Langerhans cell tumors, and thus could be useful for the diagnosis of malignancy in histiocytic neoplasms. The MAPK pathway " double hit " profile provides a basis for targeted therapy in LCS patients.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. The update of the 4th edition of the 2017 WHO classification provides new concepts in the classification of DLBCL with the definition of 2 new categories : High-grade B cell lymphoma (HGBL) with MYC, BCL2 and/or BCL6 rearrangements so-called double-hit (DH) or triple-hit (TH) lymphomas, and HGBL, not otherwise specified (NOS) without MYC and BCL2 or BCL6 translocations [1] . Patients with MYC/BCL2 DHL, MYC/BCL6 DHL or MYC/BCL2/BCL6 THL will usually have an aggressive clinical course. We report here genomic characterization of 20 adult patients diagnosed with DHL harboring MYC and BCL2 (n = 15) or MYC and BCL6 (n = 2) rearrangements and 3 THL. To identify molecular variants, we performed targeted next generation sequencing (NGS) of FFPE samples of DHL/THL for mutations on 43 genes known to be important for lymphomagenesis. In all FFPE DHL/THL cases, our NGS Lymphopanel identified 438 alterations on 40 genes. Of these, 197/438 (45%) alterations localized on 33 genes are non-synonymous and predict amino-acid substitutions or truncation of the proteins. All samples harbor non-synonymous somatic alterations (5 to 18). The most frequently mutated genes are CREBBP (16/20 cases) followed by BCL2 (12/20), KMT2D (12/20), MYC (9/20), EZH2 (8/20), IGLL5 (8/20), FOXO1 (6/20) and SOCS1 (6/20). We also assessed the impact of AID involvement and found that IGLL5, SOCS1, MYC and BCL2 have the highest AID mutation frequency (40%, 38%, 30% and 27%, respectively) whereas KMT2D, FOXO1 and EZH2 show no AID induced mutations. Furthermore, DHL/THL mutations are mainly involved in apoptosis/cell-cycle (35% of total variants) and epigenetic regulation pathways (32% of total variants), known to be associated with a poor prognosis in lymphoma patients. In conclusion, our study describes for the first time the mutational landscape of DHL/THL, a poorly studied subtype of aggressive B cell lymphoma [2] .

This study tested the sensitivity and specificity of VE1 antibody raised against BRAFV600E protein, on 189 melanoma samples, compared with molecular testing. In addition, the therapeutic response to BRAF inhibitors was analysed in 27 patients, according to staining intensity (scored from weak to strong) and pattern (homogeneous or heterogeneous). BRAFV600E status during melanoma progression was evaluated in a cohort of 54 patients with at least paired-samples. High sensitivity (98.6%) and specificity (97.7%) of VE1 were confirmed. During melanoma progression different samples showed concordant phenotypes. Heterogeneous VE1 staining was observed in 28.5% of cases, and progression-free survival was higher in patients with tumour samples displaying such staining. These findings suggest that only VE1-negative tumours would be genotyped to detect other BRAFV600 mutations, and that either primary melanoma or metastasis can be tested using immunohistochemistry, according to the material available.

Rearrangements of the anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) represent a novel molecular target in a small subset of tumors. Although ALK rearrangements are usually assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), molecular approaches have recently emerged as relevant alternatives in routine laboratories. Here, we evaluated the use of two different amplicon-based next-generation sequencing (NGS) methods (AmpliSeq and Archer®FusionPlex®) to detect ALK rearrangements, and compared these with IHC and FISH. A total of 1128 NSCLC specimens were screened using conventional analyses, and a subset of 37 (15 ALK-positive, and 22 ALK-negative) samples were selected for NGS assays. Although AmpliSeq correctly detected 25/37 (67.6%) samples, 1/37 (2.7%) and 11/37 (29.7%) specimens were discordant and uncertain, respectively, requiring further validation. In contrast, Archer®FusionPlex® accurately classified all samples and allowed the correct identification of one rare DCTN1-ALK fusion, one novel CLIP1-ALK fusion, and one novel GCC2-ALK transcript. Of particular interest, two out of three patients harboring these singular rearrangements were treated with and sensitive to crizotinib. These data show that Archer®FusionPlex® may provide an effective and accurate alternative to FISH testing for the detection of known and novel ALK rearrangements in clinical diagnostic settings.

The cytotoxic T-cell and natural killer (NK)-cell lymphomas and related disorders are important but relatively rare lymphoid neoplasms that frequently are a challenge for practicing pathologists. This selective review, based on a meeting of the International Lymphoma Study Group, briefly reviews T-cell and NK-cell development and addresses questions related to the importance of precise cell lineage (αβ-type T cell, γδ T cell, or NK cell), the implications of Epstein-Barr virus infection, the significance of anatomic location including nodal disease, and the question of further categorization of enteropathy-associated T-cell lymphomas. Finally, developments subsequent to the 2008 World Health Organization Classification, including the recognition of indolent NK-cell and T-cell disorders of the gastrointestinal tract are presented.

Mycosis fungoides (MF) represents the most common epidermotropic cutaneous T-cell lymphoma (CTCL), and tumor cells typically express a mature T-helper memory phenotype. A minority of MF patients display an unusual phenotype, which may be either CD4(-)/CD8(+) or double negative. Herein, we report a case of biopsy-proven MF in a 31-year-old woman who presented with infiltrated plaques involving photoprotected areas of the skin. Immunohistochemical study combined with confocal microscopy revealed co-expression of CD4 and CD8 in a subset of atypical T lymphocytes. To our knowledge, this is the second report of a CD4/CD8 dual-positive MF.

International audience; Plasmablastic lymphoma is a rare and aggressive diffuse large B-cell lymphoma commonly associated with Epstein-Barr virus co-infection that most often occurs in the context of human immunodeficiency virus infection. Therefore, its immune escape strategy may involve the upregulation of immune-checkpoint proteins allowing the tumor immune evasion. However, the expression of these molecules was poorly studied in this lymphoma. We have investigated 82 plasmablastic lymphoma cases of whom half were Epstein-Barr virus positive. Although they harbored similar pathological features, Epstein-Barr virus positive plasmablastic lymphomas showed a significant increase in MYC gene rearrangement and had a better 2-year event-free survival than Epstein-Barr virus negative cases (P=0.049). Immunostains for programmed cell death-1, programmed cell death-ligand 1, indole 2,3-dioxygenase and dendritic cell specific C-type lectin showed a high or moderate expression by the microenvironment cells in 60%–72% of cases, whereas CD163 was expressed in almost all cases. Tumor cells also expressed programmed cell death-1 and its ligand in 22.5% and 5% of cases, respectively. Both Epstein-Barr virus positive and negative plasmablastic lymphomas exhibited a high immune-checkpoint score showing that it involves several pathways of immune escape. However, Epstein-Barr virus positive lymphomas exhibited a higher expression of programmed cell death-1 and its ligand in both malignant cells and microenvironment as compared to Epstein-Barr virus negative cases. In conclusion, plasmablastic lymphoma expresses immune-checkpoint proteins through both malignant cells and the tumor microenvironment. The expression of programmed cell death-1 and its ligand constitutes a strong rationale for testing monoclonal antibodies in this often chemoresistant disease.

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas., Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, FIS-PI12/00202 (to J.A.M.-C.), RTICC-RD12/0036/0063 (to J.A.M-C.), RTICC-RD12/0036/0068 (to M.J.C and F.P.), RTICC-RD12/0036/0022 (to J.L.F-L.), RTICC-RD12/ 0036/0070 (to J.C.), RTICC-RD12/0036/0010 (to B.B.), RTICC- RD12/0036/0044 (to M.J.B.) and RTICC-RD12/0036/0069 (to J.M.H.R. and M.G.); by Worldwide Cancer Research project grant 15-1322 (to J.A.M.-C., Y.R.C. and M.-Q.D.); by BFU2011-30097 (to Y.R.C); by MINECO SAF2013-45787-R and Marie Curie Programme FP7-PIIF-2012-328177 (to S.R.); by the French-Spanish CITTIL project (to F.P., X.A., J.A.M.-C., C.B. and P. Brousset); by SAF2012-32810, SAF2014-57791-REDC; PIE14/00066, BIO/SA32/ 14 and CSI001U14 (to I.S.G); by FIS-ISCIII projects PI13/00160 and PI14/00025, and Fundación Inocente Inocente (to C.C.); by Deutsche Krebshilfe, Molecular Mechanisms in Malignant Lymphomas Network Project (to R.S.); by the Institut Universitaire de France (to P. Brousset); by the Broad Medical Research Program of The Eli and Edythe Broad Foundation and the Hungarian Scientific Research Fund (OTKA K108429) (to P. Balogh)

International audience

Apart from microRNAs, little is known about the regulation of expression of non-coding RNAs in cancer. We investigated whether small nucleolar RNAs (snoRNAs) accumulation displayed specific signatures in acute myeloblastic and acute lymphoblastic leukemias. Using microarrays and high-throughput quantitative PCR (qPCR), we demonstrate here that snoRNA expression patterns are negatively altered in leukemic cells compared with controls. Interestingly, a specific signature was found in acute promyelocytic leukemia (APL) with ectopic expression of SNORD112-114 snoRNAs located at the DLK1-DIO3 locus. In vitro experiments carried out on APL blasts demonstrate that transcription of these snoRNAs was lost under all-trans retinoic acid-mediated differentiation and induced by enforced expression of the PML-RARalpha fusion protein in negative leukemic cell lines. Further experiments revealed that the SNORD114-1 (14q(II-1)) variant promoted cell growth through cell cycle modulation; its expression was implicated in the G0/G1 to S phase transition mediated by the Rb/p16 pathways. This study thus reports three important observations: (1) snoRNA regulation is different in normal cells compared with cancer cells; (2) a relationship exists between a chromosomal translocation and expression of snoRNA loci; and (3) snoRNA expression can affect Rb/p16 cell cycle regulation. Taken together, these data strongly suggest that snoRNAs have a role in cancer development.

B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA, miR-125b-1, is overexpressed in B-ALL cases with the t(11;14)(q24;q32) translocation; therefore, we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting B-cell regulator of immunoglobulin heavy-chain transcription (Bright)/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was downregulated in B-ALL patients with the t(11;14)(q24;q32) translocation. Repression of Bright/ARID3a blocked differentiation and conferred a survival advantage to Ba/F3 cells under interleukin-3 starvation. In addition, overexpression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis by blockade of caspase activation by a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis.

Background In susceptible individuals, multiple events may trigger pulmonary vascular remodeling and pulmonary arterial hypertension (PAH). Human herpesvirus-8 (HHV-8), a γ-herpesvirus homologous with Epstein-Barr virus (EBV), was suggested to act as a “second hit” in the development of PAH in susceptible patients. Although there is indirect evidence from in vitro and animal studies in favor of a link between γ-herpesviruses and the pathophysiology of PAH, results remain controversial. Therefore, we investigated the presence of EBV and HHV-8 in the lungs of patients with PAH. Methods Thirty-four lungs explanted from French patients with end-stage PAH (mean age, 38 ± 14 years; 19 women) were studied. Tissue samples were incorporated into tissue microarrays. Normal lung tissues served as negative controls. Kaposi sarcoma tissue served as a positive control for HHV-8, and EBV-associated lymphoma served as a positive control for EBV. The presence of HHV-8 was investigated with immunohistochemistry and polymerase chain reaction. The presence of EBV was investigated with immunohistochemistry and in situ hybridization. Results For HHV-8, none of PAH lung samples showed a “stippling” nuclear pattern classically observed in HHV-8-positive Kaposi sarcoma lesions. When studied by polymerase chain reaction, all cases remained negative. For EBV, none of the PAH lung samples showed positive staining, whatever the technique applied. Conclusions HHV-8 and EBV cannot be detected in the lungs of patients with end-stage PAH. The role of these γ-herpesviruses in the pathophysiology of PAH is, therefore, unlikely.

International audience; The clinical management of osteosarcoma differs significantly from that of chondrosarcoma; therefore, it is extremely important to diagnose these 2 types of bone tumor accurately. In the absence of a specific marker, differential diagnosis by histochemistry is sometimes impossible, especially between chondroblastic osteosarcoma and conventional chondrosarcoma. We analyzed 165 bone sarcomas by immunohistochemical staining of tissue microarrays for expression of the galectin-1 (GAL1) lectin and by Western blot experiments. We found that GAL1 was abundant in normal human osteoblasts from benign proliferations and in osteosarcomas, including chondroblastic osteosarcomas, but not in chondrosarcomas. There was a highly significant statistical difference in the percentage of stained cells (P < 10(-4)) and in the staining intensity (P < 10(-3)) of chondroblastic osteosarcomas compared to conventional chondrosarcomas. This discriminatory potential of GAL1 staining for osteosarcoma-derived tumors was confirmed by Western blotting. We propose a diagnostic test for bone tumors that takes into account the optimal discriminative values for the percentage of cells stained and the intensity of staining. The positive and negative predictive values were 85.7% (trust interval of 63.7%-97%) and 90% (trust interval of 80%-95.9%), respectively, demonstrating the pertinence of the test. Altogether, our data indicate that GAL1 is a powerful diagnostic marker that distinguishes chondroblastic osteosarcomas from conventional chondrosarcomas.

Survivin is a member of the inhibitor of apoptosis gene family, which is also implicated in mitosis regulation. Most reports in the literature impute poor prognosis to neoplasms with overexpression of this protein. The purpose of the present study is to validate and compare the immunohistochemical reactivity of malignant lymphomas and reactive lymphoid tissue using a new mouse monoclonal antibody to Survivin produced in our laboratory, 6-78. Survivin was detected by immunohistochemistry on tissue microarrays. It was shown that the antibody anti-Survivin 6-78 reliably stains formalin-fixed, paraffin-embedded reactive and neoplastic lymphoid tissues, mostly in a nuclear pattern. We confirmed using this novel antibody that Survivin immunostaining has a tendency to be lower in reactive lymphoid tissues and low-grade B cell lymphomas than in aggressive lymphomas. This antibody may represent a useful tool for standardizing the study of the immunoexpression of Survivin in neoplasms.

The repair DNA polymerase β (Polβ), when overexpressed, plays a critical role in generating genetic instability via its interference with the genomic replication program. Up-regulation of Polβ has been reported in many tumor types that exhibit genetic aberrations, including EBV-related B-cell lymphomas. However, the mechanisms responsible for its overexpression have never been examined. Here, we report that both expression and activity of Polβ, in EBV-immortalized B cells, are induced by several natural genetic variants of LMP1, an oncoprotein associated with the vast majority of EBV-related tumors. Conversely, we found that the expression of Polβ decreased when LMP1 signaling was down-regulated by a dominant negative of LMP1 or an inhibitor of the nuclear factor-κB (NF-κB) pathway, the main transduction pathway activated by LMP1, strongly supporting a role of NF-κB in the LMP1-mediated Polβ regulation. Using electrophoretic mobility shift assay experiments from several EBV-immortalized B-cell nuclear extracts, we identified an LMP1-dependent p50/c-Rel heterodimer on a proximal κB binding site (−211 to −199nt) of the Polβ promoter. This result was correlated with a specific Polβ κB transcriptional activity. Taken together, our data enlighten a new mechanism responsible for Polβ overexpression in EBV-infected cells, mediated by LMP1 and dependent on NF-κB activation. [Cancer Res 2009;69(12):5177–85]

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

The retrospective study of 34 patients with Hodgkin lymphoma-associated hemophagocytic syndrome led us to define this association as a specific disorder. Its characteristics are male predominance (male-to-female sex ratio, 3.3:1), immunodeficiency-like histological features (lymphocyte depletion, 45% of cases; mixed cellularity Hodgkin lymphoma subtype, 40%), and strong association with Epstein-Barr virus (94%).

We selected a series of 63 primary diffuse large B-cell lymphomas (DLBCLs) of bone collected in tissue microarrays from centers in France and Brazil. These cases were classified according to the expression of antigens associated with germinal center (GC; n = 42) or non-GC (n = 21) stages of B-cell differentiation. By fluorescence in situ hybridization, we found a substantial number of cases with a rearrangement of BCL2 (9/32) and c -MYC (n = 3), whereas the PAX5, BCL6, BCL1 cyclin D1, and ALK genes were in germline configuration. It is interesting that 1 case, with a GC phenotype, showed dual BCL2 and c -MYC rearrangement. The majority of the cases with rearrangements were of the GC phenotype. These results, associated with the lack of BCL6 rearrangement, suggest that bone DLBCL represents a specific group within extranodal B-cell lymphomas.

International audience; Background: Visceral leishmaniasis (VL) is endemic in large areas of the tropics, the subtropics, and the Mediterranean basin. Besides classical VL presentation, exceptional cases of a limited form of VL have been reported. Here we describe the challenges of diagnosis and management of this intriguing entity.Case summary: A 25-year-old French Caucasian man presented with marked asthenia that had lasted 6 months and was strictly isolated except for a 2-cm left cervical lymphadenopathy. The rest of the clinical examination and extensive biological exploration were unremarkable.Histological examination of the cervical lymphadenopathy showed a reactive lymphoid hyperplasia with granulomatous organization associated with small particles in the cytoplasm of epithelioid histiocytes and giant cells evocative of Leishman-Donovan bodies. Polymerase chain reaction (PCR) performed on the tissue confirmed the presence of Leishmania donovani/infantum DNA. Direct examination of a bone marrow aspiration, together with blood and bone marrow PCR, did not find other evidence for VL. Serology for leishmaniasis was unreactive. Extensive work-up for other causes of granulomatous lymphadenitis was negative. A diagnosis of localized leishmanial lymphadenopathy was made. Intravenous liposomal amphotericin B (20 mg/kg in five infusions) was initiated and well tolerated. Asthenia disappeared promptly and the patient fully recovered.Conclusion: Localized lymph node enlargement because of leishmanial infection should be included in the differential diagnosis of lymphadenopathy of unknown origin in patients who stayed or visited, even a long time ago and for a short period, endemic areas for leishmaniasis such as the Mediterranean basin. Fine-needle aspiration cytology and/or PCR for Leishmania sp of the lymphadenopathy might contribute to the diagnosis. A low-dose liposomal amphotericin B treatment might be effective, and deserves further study.

International audience; Whole-exome sequencing demonstrates new gene mutations not previously reported in osteosarcoma; dedifferentiated osteosarcoma with MDM2 amplification does not carry any other mutations, indicating that this subtype represents a distinct entity; TP53 (and RB1) inactivation is the strongest oncogenic event in osteosarcoma development, requiring a limited number of secondary genetic mutations.Whole-genome sequencing studies have recently shown that osteosarcomas (OSs) display high rates of structural variation, i.e. they contain many somatic mutations and copy number alterations. TP53 and RB1 show recurrent somatic alterations in concordant studies, suggesting that they could be key players in bone oncogenesis. we carried out whole-genome sequencing of DNA from seven high-grade OS samples matched with normal tissue from the same patients. We confirmed the presence of genetic alterations of the TP53 (including novel unreported mutations) and RB1 genes. Most interestingly, we identified a total of 84 point mutations and 4 deletions related to 82 different genes in OS samples, of which only 15 have been previously reported. Interestingly, the number of mutated genes (ranging from 4 to 8) was lower in TP53mut cases compared with TP53wt cases (ranging from 14 to 45). This was also true for the mutated RB1 case. We also observed that a dedifferentiated OS harboring MDM2 amplification did not carry any other mutations. This study suggests that bone oncogenesis driven by TP53 or RB1 mutations occurs on a background of relative genetic stability and that the dedifferentiated OS subtype represents a clinico-pathological entity with distinct oncogenic mechanisms and thus requires different therapeutic management.

Summary Objective Human herpesvirus 8 (HHV-8) and Epstein–Barr virus (EBV) are human pathogens associated to a number of neoplasms, including tumors of the Waldeyer's ring. Both viruses have been previously detected by in situ methods in tonsils and adenoids from children. HHV-8 was found in 6.8% of the cases and EBV in about one third of the cases. As they belong to the same γ-herpesvirus subfamily and share some biological characteristics, it is of medical interest to further explore their possible relationship in the Waldeyer's ring, an issue not yet addressed in the specialized literature. The purpose of the present study is to compare the presence of EBV by in situ hybridization (ISH) in tonsils and adenoids from children up to 14 years of age in cases previously shown to be positive and negative for HHV-8. Methods Paraffin wax-embedded sections consisting of 38 tonsils and two adenoids from 40 patients were analyzed. HHV-8 was detected by ISH, using the T1-1 probe for the viral mRNA. EBV was also detected by ISH, using the EBER probe. Both probes and the detection systems were provided by Novocastra. Results HHV-8 was detected in 19 tonsils and one adenoid. The other 19 tonsils and one adenoid taken from the HHV-8-negative group were selected by pairing age and gender of patients with the HHV-8-positive group. In both groups EBV was detected in 13 cases and was negative in other 7. Conclusion Although both viruses are related in many aspects, some biological and epidemiological features differ. This is reflected in the present results, as EBV is similarly detected in the groups negative and positive for HHV-8, favoring different mechanisms of spread.

A retrospective investigation of the JAK2 V617F mutation was carried out in DNA samples from 131 bone marrow (BM) core biopsy specimens corresponding to patients with polycythemia vera (PV) (n = 31), essential thrombocythemia (ET) (n = 31), chronic idiopathic myelofibrosis (CIM) (n = 18), as well as patients with normal BM and secondary reactive hyperplasia. We used the TaqMan polymerase chain reaction single nucleotide polymorphism genotyping assay to detect the specific JAK2 mutation. This technique allowed us to detect the JAK2 V617F mutation in a population containing at least 5% of homozygous mutants. Overall, the incidence of the JAK2 V617F mutation was 87% in PV, 67% in ET, and 66% in CIM. This approach proved to be reliable and more sensitive in detecting the mutation compared with that of initial studies on different materials but similar to that of recent work with various polymerase chain reaction-based techniques. Two essential findings arose from our study. First, this technique could be carried out with DNA samples, even partially degraded, from routinely processed BM core biopsy specimens. Second, after correlation with morphological features, it turned out that the characteristics of the megakaryocytes were more specific than the mutational status of JAK2 in characterizing ET and CIM. Concerning PV, as expected, the incidence of the JAK2 mutation was higher, but the morphological criteria were misleading in some cases, strongly suggesting that the combination of both histologic and molecular data would enable the characterization of virtually all cases.

Several tyrosine kinase genes are involved in chromosomal translocations in chronic myeloproliferative disorders, but there are still uncharacterized translocations in some cases. We report two such cases corresponding to atypical chronic myeloid leukaemia with a t(8;9)(p22;p24) translocation. By fluorescence in situ hybridisation (FISH) on the corresponding metaphases with a bacterial artificial chromosome probe encompassing the janus kinase 2 (JAK2) gene at 9p24, we observed a split for both patients, suggesting that this gene was rearranged. The locus at 8p22 contains different candidate genes including the pericentriolar material 1 gene (PCM1), already implicated in reciprocal translocations. The rearrangement of the PCM1 gene was demonstrated by FISH, for both patients. By RT-PCR, we confirmed the fusion of 3' part of JAK2 with the 5' part of PCM1. Sequence analysis of the chimeric PCM1-JAK2 mRNA suggests that the putative protein displays the coiled-coil domains of PCM1 and the tyrosine kinase domain of JAK2. This new translocation identifies JAK2 as a possible therapeutic target for compounds with anti-tyrosine kinase activity.

Resume Objectif Le diagnostic des hemopathies malignes repose aujourd’hui sur l’utilisation d’un nombre croissant d’anticorps mono ou polyclonaux. La distinction entre une proliferation lymphomateuse developpee a partir de precurseurs et une proliferation composee de cellules peripheriques n’est pas toujours aisee. Cette distinction a pourtant des implications therapeutiques majeures. Dans cette etude, nous avons evalue l’apport de l’anticorps monoclonal anti-TdT dans le diagnostic et la classification de ces proliferations. Materiel et methode 13 cas ont fait l’objet d’une etude immunohistochimique : 4 lymphomes lymphoblastiques de phenotype B et T, 2 lymphomes de Burkitt, 5 leucemies aigues lymphoblastiques de phenotype B et T et 2 leucemies aigues myeloides de type myelomonoblastique. Resultats Nous avons montre que la TdT etait specifiquement exprimee par les cellules lymphomateuses immatures (lymphoblastes B ou T) alors qu’elle ne l’etait pas dans les lymphomes peripheriques comme les lymphomes de Burkitt. Enfin, si la TdT peut etre exprimee dans 30 a 40 % des leucemies myeloblastiques, ces dernieres sont aisement distinguables morphologiquement et phenotypiquement des proliferations lymphoblastiques. Conclusion L’anticorps anti-TdT est un outil de choix pour differencier les proliferations lymphoblastiques des proliferations composees de lymphocytes peripheriques et doit desormais faire partie du panel d’anticorps utilises en routine.

We investigated a series of 122 cases of small cell lung carcinomas and non-small cell lung carcinomas for the presence of several viruses that are known to be oncogenic in humans. Thus, viral genomes (DNA) and/or RNA transcripts and/or proteins of human papillomaviruses (HPV) 16, 18, 31, 33, 51, Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), human cytomegalovirus (HCMV) and simian virus 40 (SV40) were investigated on tissue sections (prepared in tissue microarrays) with different techniques of immunohistochemistry and in situ hybridisation. None of the cases displayed a single positive tumour cell for all the viruses tested whatever the technique applied. Of note, in five cases of tumours with lymphoid infiltrates, we detected scattered EBV (EBER)-positive bystander lymphocytes. In three cases, a faint nuclear staining was found with the anti-latent nuclear antigen/LANA1 (HHV-8) antibody. These cases were checked by PCR with two sets of primers (orf 26 and orf 75) and remained negative for this latter virus. Taken together, our data strongly suggest that the conventional human oncogenic viruses (HPV, EBV, HCMV, HHV-8 and SV40) are unlikely to play some role in the development of lung carcinomas..

Human cytomegalovirus (HCMV) genome and related proteins have been reported in a great proportion of malignant gliomas. However, these results are unexpected since HCMV is not known as an oncogenic virus. By immunohistochemistry (with an anti-IE1 monoclonal antibody) and in situ hybridisation (with biotinylated DNA probes) on tissue microarrays and frozen sections, we investigated a French series of central nervous system (CNS) tumours, including 97 glioblastomas. In 10 cases of glioblastoma, rare astrocyte-like cells, admixed with tumour cells, stained positively for HCMV and in one case a doubtful staining of rare cells was noticed. This may indicate a reactivation of the virus under local immunosuppression but none of the cases of CNS tumours (n=132) contained HCMV genomes and/or proteins in a significant proportion of tumour cells. Our results strongly suggest that HCMV is unlikely to be implicated in the development of human malignant gliomas, at least in French cases.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

In Hodgkin's disease (HD), both neoplastic Reed-Sternberg (RS) cells and bystander B-lymphocytes may be infected by Epstein-Barr virus (EBV). We postulated that if tumorigenic EBV strains did exist, they would be preferentially found in consistently EBV-associated tumors, such as RS cells, and differ significantly from the strains present in other, non-pathological sites of the same patients. In the present study we have compared LMP1-BNLF1 polymorphism of EBV strains infecting RS cells and B-lymphocytes in lymph nodes effected by HD on the one hand, and bystander B-lymphocytes in reactive lymph nodes on the other. It appeared that viral strains detected in HD tissues including RS cells and bystander B-lymphocytes were infected by different, but related EBV strains and were four times more polymorphic than EBV strains infecting bystander B-lymphocytes of reactive lymph nodes. The question arises as to the biological significance of these observations and the origin and chronology of multiple infections in the same patient. Since RS cells are derived from B-lymphocytes it is conceivable that the latter events could have occurred during the proliferation of bystander B-lymphocytes and their EBV episome following an antigenic stimulation.

It sometimes is difficult to diagnose leishmaniasis in tissue sections or in smears, particularly in unusual sites or if few parasites are present in the lesion. Leishmania species must be differentiated morphologically from a variety of other microorganisms, including Toxoplasma gondii, Histoplasma capsulatum, Trypanosoma cruzi, and Penicillium marneffei. We tested the value of monoclonal antibody p19-11 raised against the Leishmania homologue of receptors for activated C-kinase (LACK) as an immunohistochemical marker for amastigotes of Leishmania infantum. We evaluated a total of 117 paraffin-embedded lesions due to L infantum (92 cases), T gondii (15 cases), H capsulatum (5 cases), T cruzi (3 cases), and P marneffei (2 cases). Amastigotes of Leishmania species were detected in 92 (100%) of the leishmaniasis lesions. There were no false-positive LACK immunoreactions in any of the toxoplasmosis, histoplasmosis, trypanosomiasis, or penicilliosis specimens (0/25). We found the anti-LACK antibody p19-11 to be a highly specific and sensitive paraffin-reactive immunohistochemical marker for the confirmation or identification of Leishmania species in tissue sections.

Two cases of atypical chronic myeloid leukaemia (CML) carrying the t(4;22)(q12;q11) translocation involving the breakpoint cluster region (BCR) and platelet-derived growth factor alpha receptor (PDGFRA) genes have been recently characterized. We report a third case of atypical CML with the same translocation but with a distinct breakpoint fusing BCR exon 1 with PDGFRA exon 13. The patient had a clinical presentation of CML with progressive transformation in B-cell acute lymphoblastic leukaemia. The involvement of PDGFRA led us to treat the patient with the small organic compound imatinib mesylate/STI571 (Glivec) that blocks the ATP binding site of tyrosine kinases such as Abelson, KIT and platelet-derived growth factor receptors. The patient subsequently achieved a rapid clinical and molecular response clearly demonstrating, for the first time, that Glivec is active against PDGFRA in vivo. Therefore, our study expands the list of Glivec targets and has direct biological and also clinical implications.

Biopsies from 319 haematopoietic neoplasms were immunostained for intracellular leucocyte-specific protein 1 (LSP1) to assess its distribution and to compare its diagnostic value with that of CD45 (leucocyte common antigen: LCA). Most small B-cell neoplasms expressed LSP1, but one third of diffuse large B-cell lymphomas (DLBCL) were LSP1 negative. Among the cases with DLBCL (76 samples) tested for both LSP1 and CD45, one fifth expressed only CD45, but five samples were LSP1-positive and negative for CD45. The latter pattern was also seen in four of nine myelomas. Five out of 14 T-lymphoblastic lymphomas co-expressed LSP1 and CD45, and three cases were LSP1 negative and CD45-positive. Most peripheral T-cell lymphomas co-expressed LSP1 and CD45. All anaplastic lymphoma kinase (ALK)-negative lymphomas of anaplastic large cell morphology (T and null phenotype) expressed LSP1 although the percentage of LSP1-positive tumour cells was variable, however, only seven out of 30 cases with ALK-positive lymphoma were LSP1 positive. LSP1 was expressed on Reed-Sternberg cells in 60 out of 66 cases with classic Hodgkin's disease but neoplastic cells were usually negative in lymphocyte predominant Hodgkin's. This study confirms the wide expression of LSP1 within haematopoietic neoplasms and its diagnostic value for a minority of lymphoid tumours that have lost CD45 expression. Furthermore, the strong expression of LSP1 in classic Hodgkin's disease, contrasting with its heterogeneous expression in ALK-negative anaplastic lymphomas, may help to distinguish the latter lymphomas from patients with tumour cell-rich Hodgkin's disease.

Epstein-Barr virus (EBV) has been closely associated with undifferentiated nasopharyngeal carcinoma (NPC) and T/NK nasal non Hodgkin lymphoma. Nevertheless, the presence of EBV in non neoplastic lymphoid tissue of the nasopharynx has been rarely investigated. In a previous study by our group, using in situ hybridization to detect EBV in adenoids of children (2-13 years old) resected because of nasal obstruction due to hypertrophy, we found EBV genome in 72% of the cases. It was now intended to study the frequency of EBV expression in adenoids from children that underwent surgical removal, belonging to a lower age group (1-2 years old). It was also intended to establish which lymphoid subsets are involved in this infection. Adenoidal paraffin sections from 21 patients aged 1-2 years old (mean 1.6 years), 15 males and six females were submitted to double labeling: in situ hybridization with EBER 1/2 probes to detect EBV and immunohistochemistry to determine the lymphocyte typing of EBV-positive cells (CD20 for B-lymphocytes, CD3 for T-lymphocytes and CD56 and CD57 for NK-cells). Among 21 patients, seven showed positive lymphoid cells for EBV (33%). In almost all cases, EBV-positive cells were also CD20-positive. Some EBV-positive cells showed no labeling with any of the lymphoid markers, but in no instance they were positive for CD3, CD56 or CD57. This study confirms the preferential infection of B-lymphocytes by EBV, which in some instances can down regulate the expression of CD20.