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Purpose:The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Treg) are enriched in the AML microenvironment, but the underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFNγ released by AML cells in BM Treg induction and its impact on AML prognosis.Experimental Design:BM aspirates from patients with AML were subdivided according to IFNG expression. Gene expression profiles in INFγhigh and IFNγlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFNγ release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell cocultures. In mice, AML cells silenced for ifng expression were injected intrabone.Results:IFNγhigh AML samples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. In contrast, in patients with AML, high IFNG expression was associated with poor overall survival. Notably, IFNγ release by AML cells positively correlated with a higher BM suppressive Treg frequency. In coculture experiments, IFNγhigh AML cells modified MSC transcriptome by upregulating IFNγ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFNγ release by AML cells on MSC-derived Treg induction. In vivo, the genetic ablation of IFNγ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment.Conclusions:IFNγ release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunologic landscape toward Treg induction, contributing to an immunotolerant microenvironment.See related commentary by Ferrell and Kordasti, p. 2986

Supplementary Data from Release of IFNγ by Acute Myeloid Leukemia Cells Remodels Bone Marrow Immune Microenvironment by Inducing Regulatory T Cells

Purpose: The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Treg) are enriched in the AML microenvironment, but the underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFNγ released by AML cells in BM Treg induction and its impact on AML prognosis. Experimental Design: BM aspirates from patients with AML were subdivided according to IFNG expression. Gene expression profiles in INFγhigh and IFNγlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFNγ release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell cocultures. In mice, AML cells silenced for ifng expression were injected intrabone. Results: IFNγhigh AML samples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. In contrast, in patients with AML, high IFNG expression was associated with poor overall survival. Notably, IFNγ release by AML cells positively correlated with a higher BM suppressive Treg frequency. In coculture experiments, IFNγhigh AML cells modified MSC transcriptome by upregulating IFNγ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFNγ release by AML cells on MSC-derived Treg induction. In vivo, the genetic ablation of IFNγ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment. Conclusions: IFNγ release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunologic landscape toward Treg induction, contributing to an immunotolerant microenvironment. See related commentary by Ferrell and Kordasti, p. 2986

The differential diagnosis between lymphoplasmacytic lymphoma (LPL) and marginal zone B-cell lymphoma, particularly splenic type (SMZL), can be challenging on onset of bone marrow biopsy (BMB) since morphology and phenotype are not specific and clinical features can overlap or be mildly developed at diagnosis. The LPL-specific L265P mutation in the MYD88 gene is not available in all laboratories, and genetic aberrancies identified in SMZL (del7q, mutations of NOTCH2 and KLF2) are seldom searched in routine practice. The study aim is to investigate the potential role of myeloid nuclear differentiation antigen (MNDA) expression in this specific differential diagnosis. We report MNDA reactivity in 559 patients with small B-cell lymphoma including bone marrow biopsies from 90 LPL and 91 SMZL cases. MYD88 p.Leu265Pro mutation status was assessed and confirmed as positive in 24 of 90 LPL cases, which served as the test set. MNDA staining was negative in 23 of 24 LPL cases in the test set (96%). In the 157 remaining cases (66 LPL, 91 SMZL), which served as the validation set, the MYD88 p.Leu265Pro mutation was unavailable and MNDA was more frequently expressed in SMZL (p 0.00001). In addition, immunohistochemical features more consistent with SMZL (i.e., presence of CD23+ follicular dendritic cell meshworks, polytypic plasma cells, DBA44 reactivity) were more often present in MNDA-positive cases (statistically significant for 2 such parameters). On the widest case series so far published focusing on LPL and SMZL immunohistochemical diagnosis at onset of BMB, we demonstrated that MNDA expression significantly support the diagnosis of SMZL. This observation may be of particular help in cases where the MYD88 p.Leu265Pro mutational status and/or SMZL-related genetic aberrations are unavailable.

Although primary mediastinal (thymic) large B-cell lymphoma has been primarily studied, its precise phenotype, molecular characteristics, and histogenesis are still a matter of debate. The International Extranodal Lymphoma Study Group collected 137 such cases for extensive pathological review. Histologically, the lymphomatous growth was predominantly diffuse with fibrosis that induced compartmentalized cell aggregation. It consisted of large cells with varying degrees of nuclear polymorphism and clear to basophilic cytoplasm. On immunohistochemistry, the following phenotype was observed: CD45+, CD20+, CD79a+, PAX5/BSAP+, BOB.1+, Oct-2+, PU.1+, Bcl-2+, CD30+, HLA-DR+, MAL protein+/−, Bcl-6+/−, MUM1/IRF4+/−, CD10−/+, CD21−, CD15−, CD138−, CD68−, and CD3−. Immunoglobulins were negative both at immunohistochemistry and in situ hybridization. Molecular analysis, performed in 45 cases, showed novel findings. More than half of the cases displayed BCL-6 gene mutations, which usually occurred along with functioning somatic IgVH gene mutations and Bcl-6 and/or MUM1/IRF4 expression. The present study supports the concept that a sizable fraction of cases of this lymphoma are from activated germinal center or postgerminal center cells. However, it differs from other aggressive B-cell lymphomas in that it shows defective immunoglobulin production despite the expression of OCT-2, BOB.1, and PU.1 transcription factors and the lack of IgVH gene crippling mutations.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive molecule expressed in some subsets of normal and neoplastic cells. Mature human dendritic cells (DCs) have been shown to express IDO1, but little is known about its expression and function during DC differentiation from bone marrow hematopoietic stem/progenitor cells (HSPCs). Here, we show that during invitro differentiation along the myeloid DC lineage, CD34+ HSPCs acquire IDO1 expression, which acts in a tolerogenic manner by inducing a population of fully functional CD4+CD25+ FOXP3+ T-regulatory cells. Phenotypically, CD1a+CD14- HPSC-derived DCs expressed IDO1, langerin, CD11b, and CD1c. Cell-sorting experiments demonstrated that IDO1 expression is found in a subset of CD1a+CD14-langerin+ cells, expressing CD103, which is capable of inducing T-regulatory cells in an IDO1-dependent manner. In conclusion, DC differentiation from CD34+ HSPCs results in the expression of a functionally active IDO1 protein in CD1a+langerin+, CD103-expressing DCs. These data point toward IDO1 expression as part of a tolerogenic signature during DC development.

T-cell chronic lymphocytic leukaemia (T-CLL) has recently been reclassified under the heading of T-cell prolymphocytic leukaemia (T-PLL) because of its unfavourable clinical course, independently of the morphologic features. This rare neoplasm usually shows CD4+/CD8- phenotype. Herein we report on two cases of T-PLL with CD8 expression that correspond to a possible variant of the disease first proposed by Hui et al. in 1987. These cases presented with malignant cells showing immunophenotypic features that can be easily identified and distinguished from other peripheral T-cell leukemias. However, the total number of cases studied is inadequate for defining a discrete clinico-pathologic entity with characteristic clinical features and cytogenetical abnormalities. An international collaboration in which tissue from similar cases is referred to a central pathologist for immunophenotyping and cytogenetical study, and clinical data are centrally compiled, may assist in defining this rare malady as a discrete clinico-pathologic entity.

Diagnosis of B-non Hodgkin lymphomas (NHLs) is based on clinical, morphological and immunohistochemi-cal features. However, in up to 10-15% of cases, analysis of immunoglobulin heavy (IGH) or light (IGK/IGL) chains genes is required to discriminate between malignant and reactive lymphoid proliferations. In this study, we evaluated the feasibility and efficiency of IGK analysis in the routine diagnostic of B-cell lymphoproliferative disorders (B-LD) when applied to formalin-fixed paraffin-embedded (FFPE) tissues. Clonality patterns were studied in 59 B-LD using the BIOMED-2 protocol for IGK assays, after failure of the IGH assay. PCR products were evaluated by both heterodu-plex and GeneScan analysis. IGK analysis was technically successful in all cases. Overall, it supported the histopa-thological suspicion in 52/59 cases (88%), the sensitivity and specificity being 83% and 80%, respectively. Further, positive and negative predictive values were 95% and 50%, respectively. Interestingly, among various lymphoma subtypes, marginal zone lymphoma and follicular lymphoma most frequently required IGK analysis. In conclusion, IGK study according to the BIOMED-2 protocol resulted feasible and extremely useful in supporting challenging diagnosis of B-LD even if applied on FFPE samples. Accordingly, when NHL is suspected, negative results at IGH analysis should not be considered as conclusive and further investigation of IGK is appropriate.

Myeloid sarcoma ( MS) is a rare neoplasm whose knowledge is largely based on case reports and/or technically dated contributions. Ninety-two MSs in adulthood with clinical data available were evaluated both morphologically and immunohistochemically. Seventy-four cases were also studied by fluorescent in situ hybridization on tissue sections and/or conventional karyotyping on bone marrow or peripheral blood. Histologically, 50% of the tumors were of the blastic type, 43.5% either monoblastic or myelomonocytic and 6.5% corresponded to different histotypes. CD68/KP1 was the most commonly expressed marker (100%), followed by myeloperoxidase (83.6%), CD117 (80.4%), CD99 (54.3%), CD68/PG-M1 (51%), CD34 (43.4%), terminal-deoxy-nucleotidyl-transferase (31.5%), CD56 (13%), CD61/linker for activation of T cells (2.2%), CD30 ( 2.2%) and CD4 (1.1%). Foci of plasmacytoid monocyte differentiation were observed in intestinal cases carrying inv16. Chromosomal aberrations were detected in about 54% of cases: monosomy 7(10.8%), trisomy 8(10.4%) and mixed lineage leukemia-splitting (8.5%) were the commonest abnormalities, whereas t(8; 21) was rare ( 2.2%). The behavior was dramatic irrespective of presentation, age, sex, phenotype and cytogenetics. Most if not all, long survivors received bone-marrow transplantation. The present report expands the spectrum of our knowledge showing that MS has frequent monoblastic/myelomonocytic differentiation, displays distinctive phenotypic profile, carries chromosomal aberrations other than t( 8; 21), and requires supra-maximal therapy.

Acute myeloid leukemia cells constitutively express the immunoregulatory enzyme indoleamine 2,3-dioxygenase

PurposeAlthough peripheral T-cell lymphoma, unspecified (PTCL/U), is the most common T-cell tumor in Western countries, no study to date has been based on the application of a wide panel of markers to a large series of patients and assessed the impact of phenotype on survival. We evaluated the expression of 19 markers in 148 PTCLs/U and 45 PTCLs of the angioimmunoblastic type (AILD).Patients and MethodsThe analysis was performed on tissue microarrays by immunohistochemistry and in situ hybridization. Clinical data were available in 93 PTCL/U patients, most of whom had been included in a previous study proposing a prognostic index (PIT).ResultsAn aberrant phenotype with frequent loss of CD5 and/or CD7 was typical for PTCLs, irrespective of whether they were U or AILD. Aberrantly expressed proteins rarely included CD20, CD15, and CD30. Positivity for Epstein-Barr virus–associated small RNAs and CD15 expression emerged as adverse prognostic factors. Among PTCLs/U, the proliferation-associated protein Ki-67 turned out to be prognostically relevant and was integrated in a new predictive score, incorporating age (> 60 years), high lactate dehydrogenase, poor performance status, and Ki-67 ≥ 80%. This score was associated with the patient outcome (P < .0001) and was found to be more robust than PIT (P = .0043) in the present series.ConclusionOur retrospective analysis shows a wide range of protein expression in PTCLs and proposes a new prognostic index. The latter represents one of the first examples of mixed score (including patient- and tumor-specific factors) applied to malignant lymphomas and may be the basis for future prospective therapeutic trials.

In this study we have investigated the expression of three B-cell-associated transcription factors in normal lymphoid tissue and in T-cell neoplasms (three cell lines, and more than 50 biopsy samples). Nuclear OCT-1 immunoreactivity was seen in normal B cells, in many extrafollicular T cells, and in a heterogeneous pattern (ranging in intensity from weak to moderate) in most T-cell neoplasms. OCT-2 immunostaining was primarily restricted in normal lymphoid tissue to B cells, and was absent from most T-cell neoplasms. In contrast, immunostaining for BOB-1/OCA-B--essentially restricted to B cells in normal lymphoid tissue, with the exception of activated T-lymphocytes--was seen in all of the T-cell lines tested and the majority of the tumor cells in all categories of T-cell lymphoma. Thus labeling for each of these three B-cell-associated transcription factors can be seen to varying degrees in T-cell neoplasms. However, the high frequency of BOB-1 expression in T-cell neoplasms, in contrast to its absence from resting peripheral T cells, suggests that its expression might be a prerequisite for neoplastic transformation, and prompts a search for the transcriptional target(s) of this factor in T cells.

The expression of cytotoxic T-lymphocyte antigen-4 (CTLA-4) molecule in human normal and neoplastic hematopoietic cells, both on the cell membrane and in the intracellular compartment, was evaluated. Flow cytometric analysis carried out with a panel of anti-CTLA-4 human single-chain fragment of variable domain (scFv) antibodies revealed that CTLA-4 was not expressed on the surface, whereas it was highly expressed within the cytoplasm, in freshly isolated peripheral blood mononuclear cells (PBMCs), T cells, B cells, CD34+ stem cells, and granulocytes. Various treatments with agents able to specifically activate each cell type induced CTLA-4 expression on the surface of these cells. Similarly, increased CTLA-4 expression Was observed in different hematopoietic cell lines although they also expressed surface CTLA-4, at different degrees of intensity, before activation. Surprisingly, CTLA-4 RNA transcripts were detectable in such cell lines only after nested polymerase chain reaction (PCR) specific for CTLA-4 extracellular domain, suggesting a very fast CTLA-4 RNA processing accompanied by prolonged CTLA-4 protein accumulation. We further demonstrated surface expression of CTLA-4 in a variety of acute and chronic myeloid leukemias (AMLs and CMLs) and B- and T-lymphoid leukemias, either adult or pediatric. CTLA-4 was expressed in 25% to 85% of AMLs and CMLs depending on the leukemia subtype and the epitope analyzed, whereas in acute B-and T-leukemias CTLA-4 expression was mainly cytoplasmic. Chronic B leukemias appeared to express CTLA-4, both on the surface and in cytoplasm, whereas few cases tested of chronic T leukemias were negative. Two anti-CTLA-4 immunotoxins (scFvs-saporin) induced in vitro apoptosis of neoplastic cells from a representative AML, suggesting a novel immunotherapeutic approach to AM L based on CTLA-4 targeting. © 2003 by The American Society of Hematology.

Background/Aims: Paediatric primary follicular lymphoma of the testis (PPFLT) is exceptional: the few reported cases seem to lack BCL-2 gene rearrangement and/or protein expression. The aim of this study was to characterise a PPFLT arising in a 4 year old boy. Methods: This case was characterised using conventional histological analysis, immunohistochemistry, and a polymerase chain reaction based method for the detection of immunoglobulin V H chain rearrangements. Results: The neoplasm was staged I E /A; left orchiectomy and chemotherapy were performed, producing complete remission. Histology showed a predominantly follicular lymphoid infiltrate mainly composed of centroblast-like cells. The phenotype was CD20 + , CD79a + , CD10 + , bcl-6 + , B cell specific activating protein + , κ light chain + , CD30 −/+ , interferon regulating factor 4 −/+ , c-myc −/+ , λ light chain − , CD3 − , bcl-2 − , p53 − , cytokeratin − , and placental alkaline phosphatase − . Lymphomatous elements were found within a CD21 + follicular dendritic cell network and 70% were positive for Ki-67/MIB-1. Molecular analysis revealed monoclonal immunoglobulin heavy chain gene rearrangement and BCL-6 mutations, in the absence of BCL-2 major breakpoint and BCL-2 minor cluster region rearrangements, p53 mutations, and death associated protein kinase gene hypermethylation. Conclusions: These findings suggest a different pathogenesis of PPTFL compared with adult follicular lymphoma and might explain its favourable course in spite of aggressive histology.

Forty-five examples of Kikuchi's lymphadenitis (KL), 5 Kikuchi-like lupus erythematosus lymphadenopathies, 25 nonnecrotizing lymphadenitidies (5 toxoplasmic, 5 sarcoid-like, 6 dermatopathic, 4 suppurative, 3 tubercular, 2 with sinus histiocytosis), 4 examples of hyaline-vascular Castleman disease (CD), 2 plasmacytoid monocyte tumors (PM-Ts), and 61 accessory cell neoplasms were studied by a panel of antibodies, including the PG-M1 (against a macrophage-restricted CD68 epitope) and a polyclonal anti-myeloperoxidase (MPO). In KL and Kikuchi-like lupus erythematosus lymphadenopathies, 25 to 75% of CD68(+) histiocytes co-expressed MPO. This did not occur in nonnecrotizing lymphadenitidies and accessory cell neoplasms. MPO(+)/CD68(+) elements corresponded to nonphagocytosing mononuclear cells and some crescentic macrophages and phagocytosing histiocytes. Typical PMs were MPO(-)/CD68(+) in all cases, including CD and PM-T. Our observations suggest that in KL and KL-like lymphadenopathies: 1) MPO(+)/CD68(+) blood monocytes might be attracted into tissues because of the lack or paucity of granulocytes and the need of MPO for oxidative processes; 2) PMs are more likely to be involved in the cytotoxic immune reaction than in phagocytic phenomena; 3) the peculiar phenotype of the histiocytic component can be usefully used for the differentiation from malignant lymphoma and PM-T.

Anaplastic large cell lymphoma (ALCL) shows a wide morphologic spectrum, including the occurrence of reactive components obscuring the neoplastic population. This makes its distinction from hyperimmune reaction difficult. The authors describe an ALCL in a girl wha had a tick bite 20 days prior to clinical presentation. She developed a huge epithelioid reaction (an unprecedented finding for this tumor). The diagnostic controversies were solved by applying the ALKc antibody against anaplastic large cell lymphoma kinase (ALK), in conjunction with reagents anti-nucleophosmin (NPM), which showed the typical staining pattern observed in ALCL carrying t(2;5). Comprised within the epithelioid component there were large anaplastic cells and small-medium sized atypical elements displaying strong nuclear and cytoplasmic positivity for ALK and NPM (N-terminal region). This pattern, never observed in normal lymphocytes, corresponds to the presence of the product of the hybrid gene NPM/ALK produced by t(2;5). Following the diagnosis, the patient - whose general conditions were critical - underwent aggressive chemotherapy, achieving complete remission.

We report on a primary mediastinal large B-cell lymphoma with aberrant expression of beta-human chorionic gonadotropin (beta-hCG). The patient, a 33-year-old man, had cough, dyspnea, fever, superior vena cava syndrome, and a mediastinal bulky tumor. A biopsy showed that the latter was characterized by large cells, sclerosis, and compartmentalization. The neoplastic elements expressed CD45, CD20, CD79a and, partially, CD30, whereas they were negative for CD3, epithelial membrane antigen and cytokeratins. Surprisingly, they displayed a clear-cut positivity for beta-hCG. The remaining oncofetal markers applied (PLAP and alpha1-fetoprotein) were negative. Electron microscopy demonstrated the presence of numerous nuclear pockets and the lack of intercellular junctions. DNA analysis by polymerase chain reaction showed clonal rearrangement of Ig heavy-chain genes. The patient responded promptly to the administration of MACOP-B. To the best of our knowledge, this is the first example of B-cell lymphoma showing positivity for beta-hCG; a similar aberrant expression was previously observed only in three Japanese patients with human T-cell lymphotropic virus type I+ adult T-cell lymphoma/leukemia. Because primary mediastinal large B-cell lymphoma has in the past been frequently confused with germ cell tumors, pathologists should be aware of possible beta-hCG expression by lymphomatous cells to avoid the risk of misdiagnosis.

Immunohistochemistry of acute leukaemias in bone-marrow paraffin sections is commonly thought to be useless because of the poor preservation of many lineage-related markers. The recent development of antibodies against fixative-resistant epitopes and of new antigen retrieval techniques, however, has expanded the possibility of accurately testing routine samples. To assess the relevance of paraffin section phenotyping in lineage determination, 110 examples of acute leukaemia were studied by specific antibodies against CD1a, CD3, CD15, CD20, CD34, CD68, CD79a, TdT, myeloperoxidase, glycophorin A, and factor-VIII-related antigen. The cases included 59 acute myeloid leukaemias, classified according to the FAB cooperative group criteria, 39 precursor B-cell acute lymphoblastic leukaemias (ALLs), seven T-ALLs, and five mixed precursor B-cell/myeloid acute leukaemias. The combination of the markers employed always allowed the identification of the cell lineage (myeloid, lymphoid or mixed) and, in some instances, of phenotypic profiles characteristic of distinct acute leukaemia subtypes. According to the results obtained, bone-marrow biopsy may be regarded as a reliable tool for acute leukaemia diagnosis; this observation is of practical relevance especially for the classification of cases which lack circulating blasts in the peripheral blood or showing dry tap at bone-marrow aspiration.

In a percentage of cases, conventional morphologic evaluation of bone-marrow needle biopsy (BMNB) in insufficient to achieve a firm diagnosis. Under these circumstances, immunohistochemistry plays a basic role, providing an easy and objective key for the interpretation of the pattern observed in most instances. Herein, the authors focus on the technical procedures, which allow extensive application of immunohistochemistry to the study of BMNB, as well as on the panels of antibodies needed for the identification of the most relevant conditions.

AIM: To assess a newly developed immunohistochemical detection system, the EnVision++. METHODS: A large series of differently processed normal and pathological samples and 53 relevant monoclonal antibodies were chosen. A chessboard titration assay was used to compare the results provided by the EnVision++ system with those of the APAAP, CSA, LSAB, SABC, and ChemMate methods, when applied either manually or in a TechMate 500 immunostainer. RESULTS: With the vast majority of the antibodies, EnVision++ allowed two- to fivefold higher dilutions than the APAAP, LSAB, SABC, and ChemMate techniques, the staining intensity and percentage of expected positive cells being the same. With some critical antibodies (such as the anti-CD5), it turned out to be superior in that it achieved consistently reproducible results with differently fixed or overfixed samples. Only the CSA method, which includes tyramide based enhancement, allowed the same dilutions as the EnVision++ system, and in one instance (with the anti-cyclin D1 antibody) represented the gold standard. CONCLUSIONS: The EnVision++ is an easy to use system, which avoids the possibility of disturbing endogenous biotin and lowers the cost per test by increasing the dilutions of the primary antibodies. Being a two step procedure, it reduces both the assay time and the workload.

Summary Background One hundred sixty-eight peripheral T-cell lymphomas (PTCLs) were reviewed according to the Revised European-American Lymphoma (R.E.A.L.) Classification. Patients and methods The cases, originally diagnosed on the basis of the Updated Kiel Classification (UKC), were all provided with histological preparations, immunophenotype, clinical information, and follow-up data. The slides were reclassified by five observers, who integrated the R.E.A.L criteria with cell size measurements. The prognostic value of clinical and pathologic findings was assessed by univariate and multivariate analysis. Results The R.E.A.L. Classification was reproducibly applied by all of the observers. Clinically, anaplastic large cell lymphomas (ALCLs) differed from the remaining PTCLs by mean age (29.5 vs. 52.9 years), bulky disease (52.3% vs. 11.3%; P = 0.000), mediastinal mass (52.7% vs. 32%; P = 0.004), and disease-free survival (68.0% vs. 38.2%; P = 0.0001). Although each histological type displayed specific clinical aspects, PTCLs other than ALCL were basically characterised by a poor clinical outcome which was not influenced by the UKC malignancy grade. At multivariate analysis, the risk of a lower complete remission rate was related to bulky disease (P =0.001), histologic group (non-ALCL) (P = 0.01), and advanced stage (III–IV) (P – 0.0002). Conclusions The present study supports the classification of T-cell lymphomas proposed by the R.E.A.L. scheme.

Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed acute myeloid leukemia (AML) patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. Moreover, we demonstrated that IDO-expressing AML cells are capable to promote the differentiation of new CD4+CD25+Foxp3+ T regulatory cells (Treg cells). AML cells may be differentiated into leukemic dendritic cells (AML-DCs) which have increased immunogenicity and may be used as vaccine against leukemia. We in vitro generated DCs from 7 AML samples according to standard procedures and tested IDO expression and activity. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. Similarly to undifferentiated IDO+ AML blasts, IDO-expressing AML-DCs induced a population of CD4+CD25+Foxp3+ Treg cells, which were capable to suppress in vitro allogeneic naïve T-cell proliferation. These data identify IDO-mediated catabolism as a general tolerogenic mechanism in AML cells, including AML-DCs and raise several concerns for the use of AML-DCs as cellular vaccine against leukemia.

Acute myeloid leukemia (AML) cells may be differentiated into dendritic cells (DC) which have increased immunogenicity, but retain some immunosuppressive features of leukemic cells. Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed AML patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. We, then, in vitro generated DCs from 7 AML samples (AML-DCs) in the presence of GM-CSF, IL-4 and TNF-α. The cells we obtained were morphologically and phenotypically semi-mature DCs expressing CD40, CD80, CD86, HLA-DR and CD1a molecules and they were more efficient to induce T-cell proliferation and type 1 cytokine production than primary AML blasts. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. These data identify IDO-mediated catabolism as a tolerogenic mechanism in AML-DCs and have clinical implications for the use of AML-DCs as cellular vaccine against leukemia.

Objectives This study evaluated the role of circulating tissue factor (TF) in mediating thrombus formation on stents in an in vitro model of stent perfusion. Background The traditional view of coagulation has recently been challenged by the demonstration that TF is present in circulating blood. The potential contribution of this intravascular pool of TF to thrombus formation on stents is not known. Methods Coronary stents were placed in parallel silicone tubes connected to a roller pump that was set to pump blood at a flow rate of 10 ml/min. Stents were then exposed to heparinized blood from healthy volunteers for 120 min. Results The presence of the stent in the circuit caused a significant increase in monocyte TF expression, but only monocytes with attached platelets stained positive for TF. Thrombi formed on stents and the thrombi stained positive for TF. Pretreatment of blood with a monoclonal antibody against TF (cH36) caused a 56% reduction in 125I-fibrin(ogen) deposition on stents compared with controls (p = 0.002). Monocyte depletion of blood reduced 125I-fibrin(ogen) deposition by 45% (p = 0.01) and TF staining in the thrombus by 83% (p = 0.01). Pretreatment of blood with a monoclonal antibody against P-selectin reduced 125I-fibrin(ogen) deposition by 24% (p = 0.04). Perfusion of stents with leukocyte-reduced platelet-rich plasma (PRP) produced small thrombi and treatment of PRP with cH36 reduced 125I-fibrin(ogen) deposition by 43% (p = 0.01). Conclusions Circulating TF plays a pivotal role in thrombus formation on stents. Monocytes appear to be the main, but not only, source of TF depositing in the thrombus.

Summary Background Pyothorax-associated lymphoma (PAL) is a rare, but distinct, clinico-pathologic entity which occurs most often in Japanese people; to the best of our knowledge, only six cases of it have been reported in Western countries. The tumour develops several decades following artificial pneumothorax or chronic pleuritis due to tuberculous infection, produces pleuraleffusion associated with extensive local lymphomatous infiltrates, and is sustained by a polymorphic large B-cell clonal proliferation showing EBV integration in the genoma of the neoplastic cells. Patients and methods Herein we describe two cases of PAL observed in Italian patients, both extensively studied on the clinical, pathological, phenotypic, virological, and molecular levels. Results The two cases occurred, respectively, 45 and 50 years after therapeutic pneumothorax because of tuberculous pleuritis and were characterized by a pleural mass extending to the thoracic wall, which on histological examination were seen to consist of large elements with immunoblastic morphology. Immunohistochemistry showed monotypic restriction of Ig light chains, as well as the expression of CD45, B-cell markers (CD20, CD79a, CD45RA), bcl-2 oncogene product, EBNA-2 and, partially, LMP-1. The ratio of cycling cells was extremely high as was the number of mitotic figures. In situ hybridization displayed the presence in the neoplastic cells of the EBV related small RNAs EBER 1 and 2, which in turn, along with the positivity for EBNA-2 and LMP-1, further strengthened the close relationships between PAL and latent viral infection. Molecular studies revealed, on one hand, clonal rearrangement of the Ig heavy chain J region genes, and on the other, negativity for HHV8 in one case and positivity in the other. Conclusions These cases of PAL are the first to be documented in Italy; they serve to direct attention to the fact that this condition is not confined to Japanese people, and that its occurrence in Western countries might be underestimated.

The expression of the catalytic enzyme of tryptophan, indoleamine 2,3-dyoxigenase (IDO) in different cellular subsets, including solid tumors, has been recently identified as a T-cell inhibitory effector pathway. Here, we show that unlikely normal hematopoietic CD34+ cells expressing IDO only upon stimulation with IFN-γ, acute myeloid leukemia (AML) cells constitutively express IDO and inhibit allogeneic T-cell proliferation. IDO expression correlates with increased CD4+CD25+Foxp3+ T cells in AML patients at diagnosis. In vitro, IDO+ AML cells increase the percentage and the absolute number of CD4+CD25bright T cells, also expressing surface CTLA-4 and Foxp3 mRNA. The addition of the IDO-inhibitor, 1-methyl tryptophan (1-MT), completely abrogates such increase. Purified CD4+CD25+ T cells obtained from coculture with IDO+ AML cells are not proliferating and unable to produce IL-2. They also inhibit naive T-cell proliferation and Th1 skewing. Co-culture with IDO+ AML cells results in the conversion of CD4+CD25− into CD4+CD25+ T cells and the addition of 1-MT completely abrogates this effect. In mice, intrasplenic injection of IDO-expressing leukemia/lymphoma A20 cells induces the increase of CD4+CD25+ T cells, which can be blocked by 1-MT treatment. These data suggest that AML cells have the capacity to directly increase a population of T regulatory cells through the constitutive expression of the functionally active form of IDO. IDO expression can be regarded as a novel mechanism of leukemia escape from immune control and its inhibition may represent a novel anti-leukemia therapeutic strategy.

Routine sections of normal and pathological samples fixed in 10 per cent buffered formalin or B5, including EDTA-decalcified bone-marrow biopsies, were tested with 61 antibodies following heating in three different fluids: 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl (pH 8.0), and 1 mM EDTA-NaOH solution (pH 8.0). The sections underwent either three cycles of microwave treatment (5 min each) or pressure cooking for 1-2 min. The alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique was used as the standard detection method; with 16 antibodies a slightly modified streptavidin-biotin complex (SABC)-immunoperoxidase technique was applied in parallel. The results obtained were compared with those observed without any antigen retrieval (AR), or following section digestion with 0.05 per cent protease XIV at 37 degrees C for 5 min. Chess-board titration tests showed that all antibodies but one profited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the application of heat-based AR. By comparison with the other fluids, EDTA appeared to be superior in terms of both staining intensity and the number of marked cells. These results were independent of tissue processing, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed the simultaneous handling of a large number of slides and a time saving of 1 min 30 s, representing the proper time for the treatment.