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Minimal residual disease (MRD) monitoring plays a pivotal role in the management of hematologic malignancies. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-sequencing (RNA-seq) approach (FusionPlex) with a whole-transcriptomic strategy (Advanta RNA-Seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r = 0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection of <0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Abstract: Plasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high throughput sequencing (scATAC-seq) to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to activating protein 1 (AP-1) transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription-mediated reprogramming events that sustain PC differentiation., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma., (© 2022 by The American Society of Hematology.)

Follicular lymphoma (FL) originates in the lymph nodes (LNs) and infiltrates bone marrow (BM) early in the course of the disease. BM FL B cells are characterized by a lower cytological grade, decreased proliferation, and a specific phenotypic and subclonal profile. Mesenchymal stromal cells (MSCs) obtained from FL BM display a specific gene expression profile (GEP), including enrichment for a lymphoid stromal cell signature, and an increased capacity to sustain FL B-cell growth. However, the mechanisms triggering the formation of the medullar FL permissive stromal niche have not been identified. In the current work, we demonstrate that FL B cells produce extracellular vesicles (EVs) that can be internalized by BM-MSCs, making them more efficient to support FL B-cell survival and quiescence. Accordingly, EVs purified from FL BM plasma activate transforming growth factor β-dependent and independent pathways in BM-MSCs and modify their GEP, triggering an upregulation of factors classically associated with hematopoietic stem cell niche, including CXCL12 and angiopoietin-1. Moreover, we provide the first characterization of BM FL B-cell GEP, allowing the definition of the landscape of molecular interactions they could engage with EV-primed BM-MSCs. This work identifies FL-derived EVs as putative mediators of BM stroma polarization and supports further investigation of their clinical interest for targeting the crosstalk between BM-MSCs and malignant B cells., (© 2021 by The American Society of Hematology.)

Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK-cell expansion is challenging. We assessed the value of killer immunoglobulin-like receptor(KIR) phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (n = 46) and a reactive NK group (n = 68). We then developed an NK-cell clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the patients with CLPD-NK, constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK preferentially exhibited a CD16low phenotype, more frequently displayed a lower platelet count, and was associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed whole-exome sequencing of sorted, myeloid, T, and NK cells and found that TET2 mutations were shared by myeloid and NK cells in 3 of 4 cases. Thus, we hypothesized that TET2 alterations occur in early hematopoietic progenitors which could explain a potential link between CLPD-NK and myeloid malignancies. Finally, we analyzed the transcriptome by RNA sequencing of 7 CLPD-NK and evidenced 2 groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including 2 TET2-mutated cases, significantly underexpressed genes known to be downregulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK-cell proliferative disorders and, potentially, new therapeutic opportunities., (© 2021 by The American Society of Hematology.)

The terminal differentiation of B cells into antibody-secreting cells (ASCs) is a critical component of adaptive immune responses. However, it is a very sensitive process, and dysfunctions lead to a variety of lymphoproliferative neoplasias including germinal center-derived lymphomas. To better characterize the late genomic events that drive the ASC differentiation of human primary naive B cells, we used our in vitro differentiation system and a combination of RNA sequencing and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC sequencing). We discovered 2 mechanisms that drive human terminal B-cell differentiation. First, after an initial response to interleukin-4 (IL-4), cells that were committed to an ASC fate downregulated the CD23 marker and IL-4 signaling, whereas cells that maintained IL-4 signaling did not differentiate. Second, human CD23- cells also increased IRF4 protein to levels required for ASC differentiation, but they did that independently of the ubiquitin-mediated degradation process previously described in mice. Finally, we showed that CD23- cells carried the imprint of their previous activated B-cell status, were precursors of plasmablasts, and had a phenotype similar to that of in vivo preplasmablasts. Altogether, our results provide an unprecedented genomic characterization of the fate decision between activated B cells and plasmablasts, which provides new insights into the pathological mechanisms that drive lymphoma biology., (© 2021 by The American Society of Hematology.)

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-κB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-κB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-κB signaling, indicating a tight interplay between HSP110 and the NF-κB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-κB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-κB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL., (© 2018 by The American Society of Hematology.)

The benefit of radiotherapy (RT) after chemotherapy in limited-stage diffuse large B-cell lymphoma (DLBCL) remains controversial. We conducted a randomized trial in patients with nonbulky limited-stage DLBCL to evaluate the benefit of RT after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients were stratified according to the modified International Prognostic Index, including lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, age, and disease stage. The patients received 4 or 6 consecutive cycles of R-CHOP delivered once every 2 weeks, followed or not by RT at 40 Gy delivered 4 weeks after the last R-CHOP cycle. All patients were evaluated by fluorodeoxyglucose-positron emission tomography scans performed at baseline, after 4 cycles of R-CHOP, and at the end of treatment. The primary objective of the trial was event-free survival (EFS) from randomization. The trial randomly assigned 165 patients in the R-CHOP arm and 169 in the R-CHOP plus RT arm. In an intent-to-treat analysis with a median follow-up of 64 months, 5-year EFS was not statistically significantly different between the 2 arms, with 89% ± 2.9% in the R-CHOP arm vs 92% ± 2.4% in the R-CHOP plus RT arm (hazard ratio, 0.61; 95% confidence interval [CI], 0.3-1.2; P = .18). Overall survival was also not different at 92% (95% CI, 89.5%-94.5%) for patients assigned to R-CHOP alone and 96% (95% CI, 94.3%-97.7%) for those assigned to R-CHOP plus RT ( P = not significant). R-CHOP alone is not inferior to R-CHOP followed by RT in patients with nonbulky limited-stage DLBCL. This trial was registered at www.clinicaltrials.gov as #NCT00841945., (© 2018 by The American Society of Hematology.)

Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the accumulation of germinal center-derived malignant B cells engaged in a bidirectional crosstalk with their supportive microenvironment in invaded lymph nodes (LNs) and bone marrow (BM). T follicular helper (T FH ) cells and infiltrating stromal cells have been shown to favor FL B-cell growth, but the mechanisms of their protumoral effect and how the LN/BM microenvironment is converted into a lymphoma-permissive cell niche remain poorly understood. We demonstrated here that FL-infiltrating LN and BM stromal cells overexpressed CXCL12 in situ. Interleukin-4 high (IL-4 hi ) FL-T FH cells, unlike FL B cells themselves, triggered CXCL12 upregulation in human stromal cell precursors. In agreement, expression of CXCL12 was associated with IL-4 expression and signaling within the FL BM and LN niches. This IL-4/CXCL12 axis was amplified in activated lymphoid stromal cells as shown in our in vitro model of human lymphoid stroma differentiation and in an inducible mouse model of ectopic lymphoid organ formation. Finally, CXCL12 triggered primary FL B-cell activation, migration, and adhesion, a process antagonized by BTK and PI3K inhibitors. These data identified the IL-4/CXCL12 loop as a previously unrecognized pathway involved in lymphoid stroma polarization and as a potential therapeutic target in FL patients., (© 2017 by The American Society of Hematology.)

Introduction: The monitoring of minimal residual disease (MRD) approach in patients diagnosed with B-acute lymphoblastic leukemia (B-ALL) allows an early detection of residual clones inducing relapses and therefore appropriate therapy strategy. The molecular markers may identify and quantify the residual blasts in B-ALL with normal cytology. In this study, we aimed to use combined IKZF1, IGH and IGK immunoglobulin genes for diagnosis and MRD monitoring in B-ALL sample using MLPA, multiplex PCR and real-time quantitative PCR., Material: We showed that multiplex PCR and MLPA are necessary and complementary to detect IKZF1 deletions., Results: We have identified at the diagnosis clonal IGH rearrangement (VH3-JH5) and IKZF1 deletion (Δ4-7), which we have used it for MRD evaluation after induction chemotherapy. Despite the absence of chromosome abnormality, the patient may be classified in high-risk group with a relapse rate of residual blasts>10 -4 and sensitivity up to 10 -5 . This molecular approach enabled the patient's stratification, which was overlooked by classical methods., Conclusion: The combined IKZF1 and immunoglobulin genes will be used as appropriate molecular tools for diagnosis and MRD assessment of B-lineage leukemias and introduced as a routine tests in Tunisian clinical laboratories. They will be useful to stratify patients into risk groups leading to better treatment strategy., (Copyright © 2016 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid-derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases and in numerous tumors including multiple myeloma, chronic lymphocytic leukemia, and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified, in a cohort of 66 DLBCL patients, an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Index, event-free survival, and number of circulating Tregs. Furthermore, T-cell proliferation was restored after monocyte depletion. Myeloid-dependent T-cell suppression was attributed to a release of interleukin-10 and S100A12 and increased PD-L1 expression. In summary, we identified expanded MDSC subsets in DLBCL, as well as new mechanisms of immunosuppression in DLBCL., (© 2016 by The American Society of Hematology.)

Follicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM(+) FL B cells activated a stronger BCR signaling network than IgG(+) FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM(+) FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN-dependent adhesion of highly mannosylated IgM(+) FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN-expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets., (© 2015 by The American Society of Hematology.)

Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell-like and activated B-cell-like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation-dependent probe amplification assay and 14 gene signatures. Compared with the Affymetrix U133+2 gold standard, all 46 samples (95% CI, 92%-100%) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI, 82%-98%) of a second independent series characterized with a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable with the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from formalin-fixed, paraffin-embedded samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P = 0.01) and progression-free (P = 0.004) survival differences between the two subtypes were confirmed. Because the multiplex ligation-dependent probe amplification method is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The 2008 World Health Organization (WHO) classification distinguishes three entities among the large granular lymphocytic leukemia (LGL leukemia): T-cell LGL leukemia (T-LGL leukemia), aggressive natural killer (NK) cell leukemia, and chronic NK lymphoproliferative disorders (LPD), the later considered as a provisional entity. Only a few and small cohorts of chronic NK LPD have been published., Patients and Methods: We report here clinicobiological features collected retrospectively from 70 cases of chronic NK LPD, and compared with those of T-LGL leukemia., Results: There were no statistical differences between chronic NK LPD and T-LGL leukemia concerning median age [61 years (range 23-82 years)], organomegaly (26%), associated autoimmune diseases (24%), and associated hematological malignancies (11%). Patients with chronic NK LPD were significantly less symptomatic (49% versus 18%, P < 0.001) and the association with rheumatoid arthritis was more rarely observed (7% versus 17%, P = 0.03). The neutropenia (<0.5 × 10(9)/l) was less severe in chronic NK LPD (33% versus 61%, P < 0.001) without difference in the rate of recurrent infections. STAT3 mutation was detected in 12% of the cohort, which is lower than the frequency observed in T-LGL leukemia. Thirty-seven percent of the patients required specific therapy. Good results were obtained with cyclophosphamide. Overall and complete response rates were, respectively, 69% and 56%. Overall survival was 94% at 5 years., Conclusion: This study suggests very high similarities between chronic NK LPD and T-LGL leukemias. Since chronic NK LPD is still a provisional entity, our findings should be helpful when considering further revisions of the WHO classification., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

With intensified pediatric-like therapy and genetic disease dissection, the field of adult acute lymphoblastic leukemia (ALL) has evolved recently. In this new context, we aimed to reassess the value of conventional risk factors with regard to new genetic alterations and early response to therapy, as assessed by immunoglobulin/T-cell receptor minimal residual disease (MRD) levels. The study was performed in 423 younger adults with Philadelphia chromosome-negative ALL in first remission (265 B-cell precursor [BCP] and 158 T-cell ALL), with cumulative incidence of relapse (CIR) as the primary end point. In addition to conventional risk factors, the most frequent currently available genetic alterations were included in the analysis. A higher specific hazard of relapse was independently associated with postinduction MRD level ≥10(-4) and unfavorable genetic characteristics (ie, MLL gene rearrangement or focal IKZF1 gene deletion in BCP-ALL and no NOTCH1/FBXW7 mutation and/or N/K-RAS mutation and/or PTEN gene alteration in T-cell ALL). These 2 factors allowed definition of a new risk classification that is strongly associated with higher CIR and shorter relapse-free and overall survival. These results indicate that genetic abnormalities are important predictors of outcome in adult ALL not fully recapitulated by early response to therapy. Patients included in this study were treated in the multicenter GRAALL-2003 and GRAALL-2005 trials. Both trials were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively., (© 2014 by The American Society of Hematology.)

Antigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.

Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of cancers. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and BM. In addition, mesenchymal stromal cells (MSCs) support in vitro FL B-cell survival, in particular after their engagement toward lymphoid differentiation. We show here that BM-MSCs obtained from patients with FL (FL-MSCs) display a specific gene expression profile compared with MSCs obtained from healthy age-matched donors (HD-MSCs). This FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 could be detected at a high level within the FL-cell niche, is up-regulated in HD-MSCs by coculture with malignant B cells, and is overexpressed by FL-MSCs, in agreement with their capacity to recruit monocytes more efficiently than HD-MSCs. Moreover, FL-MSCs and macrophages cooperate to sustain malignant B-cell growth, whereas FL-MSCs drive monocyte differentiation toward a proangiogenic and lipopolysaccharide-unresponsive phenotype close to that of tumor-associated macrophages. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL-cell niche, thus emerging as a potential therapeutic target in this disease.

It has become increasingly clear that proliferation and survival in FL is not only driven by genetic changes, but also and possibly even predominantly by the close interaction with the immune microenvironment and stromal cells. Based on in vitro studies and experimental models and supported by immunohistochemical studies in biopsy specimens of FL patients, classes of CD4+ T-cell populations including follicular helper T cells and regulatory T cells are now identified as major players to regulate the delicate balance of effector populations into a supportive microenvironment. These insights may thoroughly change the therapeutic approaches in FL and translate into programs that combine direct cytotoxic and indirect immunomodulatory aspects., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Accumulating evidence indicates that the cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LNs) and in infiltrated bone marrow where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRCs) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRCs and their postulated progenitors, that is, bone marrow mesenchymal stem cells (MSCs), in FL remains unexplored. In this study, we investigated the relationships between FRCs and MSCs and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain MSCs able to give rise to adipocytes, chondrocytes, osteoblasts, as well as fully functional B-cell supportive FRCs. In vitro, bone marrow-derived MSCs acquire a complete FRC phenotype in response to a combination of tumor necrosis factor-alpha and lymphotoxin-alpha1beta2. Moreover, MSCs recruit primary FL cells that, in turn, trigger their differentiation into FRCs, making them able to support malignant B-cell survival. Altogether, these new insights into the cross talk between lymphoma cells and their microenvironment could offer original therapeutic strategies.

Cigarette smoking, a well-known cardiovascular risk factor, has been recently demonstrated to decrease circulating endothelial progenitor cell (EPC) number. Owing to the fact that polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) constitute major components of tobacco smoke, the present study was designed to analyze the effects of these chemicals on the development of human EPC cultures from peripheral blood mononuclear cells. Treatment by BP markedly impaired EPC number and EPC colonies in a dose-dependent manner. Such deleterious effects were abrogated using 3'-methoxy-4'-nitroflavone, a pure antagonist of the aryl hydrocarbon receptor, highlighting the involvement of this receptor in PAH toxicity towards EPCs. Additional events such as cytochrome P-450-dependent PAH metabolism and formation of PAH-related adducts to cellular macromolecules were also required. Overall, these data established EPCs as new cellular targets of PAHs, which may contribute to the deleterious cardiovascular effects of environmental substances containing these chemicals, especially tobacco smoke.

The paired box containing gene PAX-5 encodes the transcription factor BSAP (B-cell-specific activator protein), which plays a key role in B-lymphocyte development. Despite its known involvement in a rare subtype of non-Hodgkin's lymphoma (NHL), a detailed examination of BSAP expression in NHL has not been previously reported. In this study, we analyzed normal and malignant lymphoid tissues and cell lines, including 102 cases of B-cell NHL, 23 cases of T- and null-cell NHL, and 18 cases of Hodgkin's disease. Normal lymphoid tissues showed strong nuclear BSAP expression in mantle zone B cells, less intense reactivity in follicular center B cells, and no expression in cells of the T-cell-rich zones. Monocytoid B cells showed weak expression, whereas plasma cells and extrafollicular large transformed B cells were negative. Of the 102 B-cell NHLs, 83 (81%) demonstrated BSAP expression. All of the 13 (100%) B-cell chronic lymphocytic leukemias (B-CLLs), 21 of (100%) mantle cells (MCLs), and 20 of 21 (95%) follicular lymphomas (FLs) were positive. Moderate staining intensities were found in most B-CLL and FL cases, whereas most MCLs showed strong reactions, paralleling the strong reactivity of nonmalignant mantle cells. Eight of 12 (67%) marginal zone lymphoma cases showed negative or low BSAP levels, and 17 of 24 (71%) large B-cell lymphomas displayed moderate to strong expression. None of the 23 T- and null-cell lymphomas reacted with the BSAP antisera, whereas in Hodgkin's disease, 2 of 4 (50%) nodular lymphocytic predominance and 5 of 14 (36%) classical cases showed weak nuclear or nucleolar BSAP reactions in a fraction of the tumor cells. Western blot analysis showed a 52-kD BSAP band in B-cell lines, but not in non-B-cell or plasma cell lines. We conclude that BSAP expression is largely restricted to lymphomas of B-cell lineage and that BSAP expression varies in B-cell subsets and subtypes of B-cell NHL. The high levels of BSAP, especially those found in large-cell lymphomas and in some follicular lymphomas, may be a consequence of deregulated gene expression and suggest a possible involvement of PAX-5 in certain B-cell malignancies. This is a US government work. There are no restrictions on its use.

Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown, in vitro studies suggest that abnormalities of the p53 gene, bcl-2 overexpression, and low tumor proliferation rates may increase chemotherapy resistance. We analyzed tumor tissue from 75 patients with relapsed/refractory NHL (Working Formulation A through H) for p53 mutation/overexpression (abnormality), bcl-2 expression, and tumor proliferation and correlated them with multiple clinical characteristics, response to therapy, disease-free survival, and overall survival (OS). All tumor biopsy specimens were obtained within 6 weeks of treatment with EPOCH (infusional etoposide, vincristine, and doxorubicin and bolus prednisone and cyclophosphamide) chemotherapy. Overall, 16 (21%) tumors had a p53 abnormality. Of 13 tumors with overexpression, mutations were confirmed by sequence analysis in 11, and, in 44 tumors without overexpression, 3 showed mutations. A multivariate analysis showed that tumors with a p53 abnormality were more likely to be drug resistant than tumors with normal p53 (56% v 17%; P2 = .008) and to have a shorter median progression-free survival (PFS; 2.1 v 8.2 months; P2 = .008) and OS (11.7 v 21.5 months; P2 = .038), respectively. The presence of a p53 abnormality did not correlate with any clinical characteristic, bcl-2 expression, or tumor proliferation. A significant correlation was found between low tumor proliferation and drug resistance in a univariate (P2 < .006) but not multivariate analysis. Patients with tumor proliferation of less than 80% were significantly more likely to have no response to therapy (31% v 6%) or to fail to achieve a complete response (16% v 44%) and tended to have shorter PFS and OS than did patients with higher proliferation. No significant association was found between bcl-2 expression and drug resistance, PFS or OS, although patients with intermediate-grade histologies and high bcl-2 expression tended to be drug resistant as compared with low level expressors (P2 < .065). Of interest was the finding of a significant association between high bcl-2 and low tumor proliferation (P2 = .0045). In studies that have found an association between high bcl-2 expression and short PFS, bcl-2 may have been a surrogate for low tumor proliferation. Further studies are warranted to examine this question. These results suggest that p53 mutation and low tumor proliferation, but not bcl-2, may be important causes of clinical drug resistance in NHL.

We have evaluated the expression and distribution of the cellular apoptosis susceptibility (CAS) protein in normal lymphoid tissue and malignant lymphomas. CAS protein, the product of the CAS gene, is associated with microtubules and the mitotic spindle. Immunohistochemistry with an antibody to CAS shows many CAS-positive cells in normal tonsils. The majority of strongly CAS-positive cells were localized to the dark zone of the follicles, whereas the mantle zone and interfollicular areas were essentially negative. Double staining for CAS and Ki-67 revealed co-expression of the two proliferation markers in approximately 85 to 90% of the CAS-positive cells. Different subtypes of lymphomas exhibited varying patterns of CAS expression. Low-grade non-Hodgkin's lymphoma generally revealed weak staining with CAS, with 10 to 60% of all cells being positive. In contrast, highly malignant non-Hodgkin's lymphoma and malignant cells of Hodgkin's disease displayed very strong CAS positivity, with staining of up to 80% of the atypical cells. Overall, the staining pattern of CAS and Ki-67 was superimposable within a particular lymphoma subtype. However, in all lymphomas we observed a significant fraction of CAS-positive normal and malignant lymphocytes that were Ki-67 negative, probably because they were momentarily noncycling cells. We conclude that a high expression of CAS correlates with proliferation of normal and malignant lymphoid cells. The fact that detection of CAS protein identifies a higher portion of proliferating and malignant cells than Ki-67 warrants further evaluation of CAS protein as a marker with a diagnostic potential.

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD-1 gene overexpression. Some aggressive variants have been recognized with a blastic or large cell morphology, higher proliferative activity, and shorter survival. p53 gene mutations in lymphoid neoplasms have been detected mainly in high grade lymphomas and have been associated with tumor progression in follicular and small lymphocytic lymphomas. To determine the role of p53 alterations in MCL, we examined 35 typical and 8 aggressive variants (5 blastic and 3 large cell) of MCLs by a combination of immunohistochemistry, single-strand conformational polymorphism analysis of genomic DNA and/or cDNA obtained by reverse transcriptase-polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. Of the 8 aggressive MCLs, 3 (38%) contained missense point mutations in axon 8 codon 278 (Pro --> Leu), exon 8 codon 273 Arg --> His), and exon 5 codon 151 (Pro --> Ser), respectively. A diffuse p53 protein overexpression was observed in more than 50% of the tumor cells in these 3 cases. A fourth blastic MCL also showed strong p53 immunoreactivity. However, no mutations were detected in exons 5-9 in this case. p53 expression was also detected in 10% of the cells in an additional large cell type of MCL and in less than 1% of the cells in 6 typical cases. No mutations were detected in any of these cases or in the remaining cases with no expression of the protein. Four nucleotide changes were observed by single-strand conformational polymorphism analysis in 4 typical MCLs with no overexpression of the protein. Direct sequencing showed that these nucleotide changes were located at exon 6 (1 case), intron 7 (2 cases), and intron 8 (1 case). The changes in exon 6 and intron 7 were known polymorphisms. The nucleotide change in intron 8 was outside splicing sites of the neighboring exons. The overall survival of the 3 patients with p53 mutations (median, 18.3 months) was significantly shorter than that of patients with the nonmutated MCLs (median, 49 months; P < .01). These findings indicate that p53 gene mutations are an infrequent phenomenon in MCLs and are associated with a subset of aggressive variants.

Primary and secondary amyloidosis are not uncommon in aging but the diagnosis is rarely made on account of the risk of bleeding in the site of biopsies and the difficulty to distinguish senile from systemic amyloidosis on the biopsy samples. We have studied the frequency of amyloid deposition in the abdominal fat aspirate (AFA), the labial salivary gland (LSG), the temporal arteries (four cases), bone marrow (two cases), digestive tract (four cases) in 100 elderly patients (aged 80 or greater). AFA was positive in 15 percent of the patients and LSG in 5%; both samples were positive in 4%. Four cases of systemic amyloidosis were found (two of the AL and two of the AA type). Sensitivity of AFA was 75%, specificity was 87% and the positive predictive value was 20%. The values were respectively 100%, 99%, 100% for LSG. In 11 patients whose AFA biopsies samples were singly positive, amyloid deposits were found in temporal arteries in four of four cases. We conclude that AFA is too sensitive for the diagnosis of systemic amyloidosis in aging. The responsibility of senile amyloid deposition on AFA should require further investigations. LSG biopsies seem to be a more reliable test for the diagnosis of primary and secondary amyloidosis in elderly.

Stauffer's syndrome is characterized by a cholestasis without biliary obstruction or hepatic metastasis and a renal tumor. We report a case of Stauffer's syndrome in a 73 year-old woman. Cholestasis and inflammatory syndrome regressed with corticosteroid. Giant cell arteritis with negative temporal artery biopsy was wrongly suspected.

We report a case of a 35 year-old-man with dermatomyositis associated with tracheopathia osteoplastica. The swallowing perturbation secondary to myositis and airway involvement by tracheopathia induced fatal outcome. Tracheopathia osteoplastica is a rare disease and occurs exclusively in men over the age of 50. The association of two rarest disease is not a fortuitous event. The common pathogenic factor may be, in this case, the occupational exposure to silicon.

Angioimmunoblastic lymphadenopathy (AIL) still is a clinico-pathological syndrome with little known physiopathology. The advent of molecular biology has improved our understanding of this syndrome by characterization of the clonal cell. With this technique, combined with cytogenetics and immunohistochemistry, three pathological states have been individualized: 1) true AIL without evidence of monoclonal proliferation; 2) transformed AIL, and 3) AIL-like T-cell lymphoma. This clinical complex can be integrated in an evolutive continuum, starting with simple lymphoid hyperplasia and ending with frank malignant T-cell lymphoma.