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Simple Summary: The aim of this study was to understand if the presence of a RHOA-G17V mutation in angioimmunoblastic T-cell lymphoma is responsible for the typical clinical presentation with early dissemination of tumor cells. Overexpression of RHOA-G17V in malignant T cells did not enhance their migration capacity neither in microfluidics microchannels, 3D collagen gel or primary human lymphoid tissue. In contrast, RHOA-G17V overexpressing cells from the HH cell line presented increased numbers of cells surrounded by cleaved collagen. Primary AITL cases showed a correlation between RHOA-G17V mutation allele frequency and collagen fibrosis of the tissue, suggesting that mechanisms leading to extracellular matrix degradation contribute to the early spread of AITL tumor cells. Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (AITL), is characterized by constitutional symptoms, advanced-stage disease, and generalized lymphadenopathy. A genetic hallmark of this lymphoma is the frequent occurrence of the RHOA mutation G17V in neoplastic cells, which is observed in around 60% of patients. Because RHOA is involved in both T-cell receptor downstream signalling and cell migration, we hypothesized that the characteristic presentation of AITL could be the result of enhanced tumor cell migration. Therefore, this study aimed to elucidate the impact of the RHOA variant G17V on the migration of neoplastic T cells. We transfected the T-cell lymphoma cell lines HH and HuT78 to stably express the RHOA-G17V variant. RHOA-G17V-expressing T cells did not exhibit enhanced motility compared to empty-vector-transfected cells in microchannels, a 3D collagen gel, or primary human lymphatic tissue. Cells of the HH cell line expressing RHOA-G17V had an increased number of cells with cleaved collagen compared with the empty-vector-transfected cells. Therefore, we hypothesized that the early spread of AITL tumor cells may be related to remodelling of the extracellular matrix. Accordingly, we observed a significant negative correlation between the relative area of collagen in histological sections from 18 primary AITL and the allele frequency of the RHOA-G17V mutation. In conclusion, our results suggest that the characteristic presentation of AITL with early, widespread dissemination of lymphoma cells is not the result of an enhanced migration capacity due to the RHOA-G17V mutation; instead, this feature may rather be related to extracellular matrix remodelling. [ABSTRACT FROM AUTHOR]

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αβ sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4+ and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4+ and CD8+ T cells. Degrees of clonal T cell expansion varied between patients (range: 1–18 expanded clones per patient) and was almost exclusively restricted to CD8+ T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells. [ABSTRACT FROM AUTHOR]

Nodular lymphocyte predominant Hodgkin lymphoma ( NLPHL) and T cell/histiocyte rich large B cell lymphoma ( THRLBCL) usually affect middle-aged men, show tumour cells with a B cell phenotype and a low tumour cell content. Whereas the clinical behaviour of NLPHL is indolent, THRLBCL presents with advanced stage disease and an aggressive behaviour. In the present study, array comparative genomic hybridization was performed in seven typical NLPHL, four THRLBCL-like NLPHL variants, six THRLBCL and four diffuse large B cell lymphomas ( DLBCL) derived from NLPHL. The number of genomic aberrations was higher in THRLBCL compared with typical and THRLBCL-like variant of NLPHL. Gains of 2p16.1 and losses of 2p11.2 and 9p11.2 were commonly observed in typical and THRLBCL-like variants of NLPHL as well as THRLBCL. Gains of 2p16.1, affecting the REL locus were confirmed in an independent cohort. Expression of the REL protein was observed at similar frequencies in typical and THRLBCL-like variant of NLPHL as well as THRLBCL (33-38%). In conclusion, the present study reveals further similarities between NLPHL and THRLBCL on the genomic level, confirming that these entities are part of a pathobiological spectrum with common molecular features, but varying clinical presentations. [ABSTRACT FROM AUTHOR]

In contrast to the commonly indolent clinical behavior of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), T cell/histiocyte rich large B cell lymphoma (THRLBCL) is frequently diagnosed in advanced clinical stages and has a poor prognosis. Besides the different clinical presentations of these lymphoma entities, there are variants of NLPHL with considerable histopathologic overlap compared to THRLBCL. Especially THRLBCL-like NLPHL, a diffuse form of NLPHL, often presents a histopathologic pattern similar to THRLBCL, suggesting a close relationship between both lymphoma entities. To corroborate this hypothesis, we performed gene expression profiling of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. In unsupervised analyses, the lymphomas did not cluster according to their entity. Moreover, even in supervised analyses, very few consistently differentially expressed transcripts were found, and for these genes the extent of differential expression was only moderate. Hence, there are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. Based on the gene expression studies, we identified BAT3/BAG6, HIGD1A, and FAT10/UBD as immunohistochemical markers expressed in the tumor cells of all three lymphomas. Characterization of the tumor microenvironment for infiltrating T cells and histiocytes revealed significant differences in the cellular composition between typical NLPHL and THRLBCL cases. However, THRLBCL-like NLPHL presented a histopathologic pattern more related to THRLBCL than NLPHL. In conclusion, NLPHL and THRLBCL may represent a spectrum of the same disease. The different clinical behavior of these lymphomas may be strongly influenced by differences in the lymphoma microenvironment, possibly related to the immune status of the patient at the timepoint of diagnosis. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation. [ABSTRACT FROM AUTHOR]

Hartmann S, Agostinelli C, Klapper W, Korkolopoulou P, Koch K, Marafioti T, Piccaluga P P, Patsouris E, Pileri S & Hansmann M-L (2011) Histopathology 59, 1173-1182 Revising the historical collection of epithelioid cell-rich lymphomas of the Kiel Lymph Node Registry: what is Lennert's lymphoma nowadays? Aims: Lennert's lymphoma is a rare variant of peripheral T-cell lymphoma (PTCL) not otherwise specified (NOS). The aim of this study was to further characterize this tumour. Methods and results: Historical material of 97 lymphomas with a high content of epithelioid cells, collected at the Kiel Lymph Node Registry were reviewed, by applying immunohistochemistry and current diagnostic criteria. Among all cases revised, various B-cell lymphoma entities (25 cases), Hodgkin lymphomas (21 cases) and PTCL subtypes (48 cases) could be identified. A distinctive subgroup of eight PTCLs was found that were regarded as genuine Lennert's lymphomas. These cases were characterized by mild atypia, a non-activated cytotoxic phenotype [TIA1 cytotoxic granule-associated RNA binding protein (TIA1)-positive+ and granzyme B-negative], and a substantial lack of follicular T-helper (TFH) cell markers. Among the other PTCLs, including angioimmunoblastic T-cell lymphoma and PTCL NOS, many cases with positivity for more than three TFH cell-associated molecules were recorded. Conclusions: Our study shows that, according to current criteria, Lennert's lymphoma is a rare but distinctive entity among epithelioid cell-rich lymphomas, differing on grounds of morphology and immunophenotype from other PTCL subtypes. An additional finding is the broad morphological spectrum of epithelioid-cell rich PTCLs showing a TFH cell phenotype. [ABSTRACT FROM AUTHOR]

Agostinelli C, Hartmann S, Klapper W, Korkolopoulou P, Righi S, Marafioti T, Piccaluga P P, Patsouris E, Hansmann M-L, Lennert K & Pileri S A (2011) Histopathology 59, 679-691 Peripheral T cell lymphomas with follicular T helper phenotype: a new basket or a distinct entity? Revising Karl Lennert's personal archive Aims: To revise 25 cases selected from Karl Lennert's personal archive (21) and Bologna and Frankfurt Registries (four) because of cytological similarities. Methods and results: All cases were provided with paraffin blocks and studied by immunohistochemistry and molecular techniques. While phenotyping was very informative, among molecular studies only EBER in situ-hybridization (ISH) was successful. Twenty-two cases were concluded as peripheral T cell lymphomas (PTCL). Of these, six were reclassified as angioimmunoblastic T cell lymphoma (AITL), 13 as PTCL, not otherwise specified (NOS), including four follicular variants and one tumour with T-zone pattern, and three as borderline tumours between AITL and PTCL/NOS. All these cases consisted homogeneously of small/medium-sized elements with mild nuclear atypia and an evident rim of clear/pale cytoplasm. On immunohistochemistry, they regularly expressed three to six follicular helper T cell (FTH)-associated markers. EBER-ISH revealed scattered EBV-infected B cells in all tumours except those with 'follicular' growth pattern. The content of follicular dendritic cells and high-endothelial venules varied significantly depending on the histotype. Conclusions: This study shows that: (i) historical material can be still employed usefully, and (ii) the FTH-phenotype corresponds to a broad spectrum of PTCLs that might form a new category to be validated in future molecular and clinicopathological analyses. [ABSTRACT FROM AUTHOR]

Little is known about genomic aberrations in peripheral T cell lymphoma, not otherwise specified (PTCL NOS). We studied 47 PTCL NOS by 250k GeneChip single nucleotide polymorphism arrays and detected genomic imbalances in 22 of the cases. Recurrent gains and losses were identified, including gains of chromosome regions 1q32–43, 2p15–16, 7, 8q24, 11q14–25, 17q11–21 and 21q11–21 (≥5 cases each) as well as losses of chromosome regions 1p35–36, 5q33, 6p22, 6q16, 6q21–22, 8p21–23, 9p21, 10p11–12, 10q11–22, 10q25–26, 13q14, 15q24, 16q22, 16q24, 17p11, 17p13 and Xp22 (≥4 cases each). Genomic imbalances affected several regions containing members of nuclear factor-kappaB signalling and genes involved in cell cycle control. Gains of 2p15–16 were confirmed in each of three cases analysed by fluorescence in situ hybridization (FISH) and were associated with breakpoints at the REL locus in two of these cases. Three additional cases with gains of the REL locus were detected by FISH among 18 further PTCL NOS. Five of 27 PTCL NOS investigated showed nuclear expression of the REL protein by immunohistochemistry, partly associated with genomic gains of the REL locus. Therefore, in a subgroup of PTCL NOS gains/rearrangements of REL and expression of REL protein may be of pathogenetic relevance. [ABSTRACT FROM AUTHOR]

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of geneassociated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes-DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1-that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs. [ABSTRACT FROM AUTHOR]

T-cell lymphomas are highly heterogeneous and their prognosis is poor under the currently available therapies. Enhancers of zeste homologue 1 and 2 (EZH1/2) are histone H3 lysine-27 trimethyltransferases (H3K27me3). Despite the rapid development of new drugs inhibiting EZH2 and/or EZH1, the molecular interplay of these proteins and the impact on disease progression and prognosis of patients with T-cell lymphomas remains insufficiently understood. In this study, EZH1/2 mutation status was evaluated in 33 monomorphic epitheliotropic intestinal T-cell lymphomas by next generation sequencing and EZH1/2 and H3K27me3 protein expression levels were detected by immunohistochemistry in 46 T-cell lymphomas. Correlations with clinicopathologic features were analyzed and survival curves generated. No EZH1 mutations and one (3%) EZH2 missense mutation were identified. In univariable analysis, high EZH1 expression was associated with an improved overall survival (OS) and progression-free survival (PFS) whereas high EZH2 and H3K27me3 expression were associated with poorer OS and PFS. Multivariable analysis revealed EZH1 (hazard ratio (HR) = 0.183; 95% confidence interval (CI): 0.044–0.767; p = 0.020;) and EZH2 (HR = 8.245; 95% CI: 1.898–35.826; p = 0.005) to be independent, divergent prognostic markers for OS. In conclusion, EZH1/2 protein expression had opposing effects on the prognosis of T-cell lymphoma patients. [ABSTRACT FROM AUTHOR]

Nodular lymphoid lesion (NLL) of the liver is a rare but unique entity and has also been termed reactive lymphoid hyperplasia of the liver. We describe the histological, immunohistochemical and molecular biologic findings of a case with NLL and two other tumors of the liver. The nodular lymphoid mass found in the liver was composed of heterogeneous small lymphocytes forming reactive follicles. Plasma cells, few immunoblasts, centroblasts, few macrophages, epithelioid cells, and giant cells were seen. The lymphoid infiltrate displaced the adjacent hepatic parenchyma. By immunohistochemistry and molecular studies, the lymphocytes were found to be polyclonal. The diagnosis of NLL was made. In addition to NLL, focal nodular hyperplasia and hemangioma were detected. The discrimination of NLL from primary hepatic malignant non-Hodgkin's lymphoma of mucosa-associated lymphoid tissue-type may pose diagnostic difficulties and may require the use of immunohistochemical and molecular techniques. The simultaneous occurrence of NLL with focal nodular hyperplasia and hemangioma in the liver has not been described before. [ABSTRACT FROM AUTHOR]

Stimulation of lymphoid cells via their surface receptors triggers signalling pathways that terminate in the nucleus, where they induce alterations in gene transcription. Nuclear factor of activated T cells (NFAT) transcription factors, involved in a major Ca2+-dependent signalling pathway, normally reside in the cytoplasm but re-locate to the nucleus when activation of the pathway (e.g. following ligation of antigen receptors) leads to their dephosphorylation. This study found that one member of the NFAT family (NFATc1/NFAT2) can be detected in routine biopsy samples, where it is seen in essentially all lymphoid cells, but is absent from the great majority of non-haematopoietic cells. An immunohistological evaluation of NFATc1 in almost 300 lymphomas showed that most neoplastic lymphoid cells also express NFATc1 as a cytoplasmic constituent, although it is absent in classical Hodgkin's disease and plasma cell proliferations. Of particular interest was the finding that NFATc1 was relocated to the nucleus in a minority of lymphoid neoplasms (usually diffuse large B-cell lymphomas or Burkitt lymphoma), presumably reflecting activation of the NFAT pathway. It would be of interest to correlate this feature with patterns of gene expression and also with prognosis, since it may identify a subset of human lymphoma that is distinct in its molecular and clinical features. [ABSTRACT FROM AUTHOR]

The significance of T-cell proliferations in angioimmunoblastic lymphoma (AILD) is still enigmatic. Although classified as a malignant T-cell lymphoma in the World Health Organisation lymphoma classification, some cases of AILD lack dominant T-cell clones. In a previous study, based on single-cell polymerase chain reaction (PCR), we obtained similar results as studies of AILD using Southern blot or conventional PCR: some cases of AILD contained large T-cell clones, and, in other cases, T-cell clones were undetectable. As in single-cell studies, only a limited number of cells could be investigated; thus, we wanted to gain more insight into the amount and distribution of tumour cells. By applying triple immunofluorescent staining with antibodies directed against T-cell receptor Vbeta-family-specific epitopes, we investigated T-cell populations in AILD and their localisation in the tissue in relation to B cells (CD20) and follicular dendritic cells (CD21). In two of five cases investigated, only a minority of the T-cells compartment belonged to the tumour clone. Neoplastic T cells were found throughout the tissue, including areas dominated by B cells. [ABSTRACT FROM AUTHOR]

We have investigated whether intracellular signal transduction molecules can be used as immunohistological markers of normal and neoplastic human leucocytes in routine tissue sections. We obtained selective labelling of white cells for eight such molecules (the ‘linker’ molecules SLP-76 and BLNK, the Src family kinases Lyn, Fyn, Syk and Hck, and the phospholipases PLC- γ1 and PLC- γ2). Antibodies to SLP-76 and PLC- γ1 selectively labelled T cells, and antibodies to BLNK, Lyn, Fyn, Syk and PLC- γ2 labelled B cells (although Fyn immunostaining was restricted to mantle zone B cells). Antibodies to the Syk and Hck kinases labelled probable thymocyte precursors at the periphery of the thymic cortex. In addition to lymphoid cells, several other leucocyte types were immunostained (e.g. SLP-76, Lyn, Syk and Hck were found in megakaryocytes, myeloid cells and/or macrophages, and PLC- γ2 was detected in arterial endothelium). SLP-76 and PLC- γ1 were found in most T-cell lymphomas studied, and some B-cell lymphomas were also positive for PLC- γ1 (e.g. diffuse large cell and Burkitt's lymphoma). The five B cell-associated markers were found in most B-cell non-Hodgkin's lymphomas, although some diffuse large B-cell lymphomas were negative (e.g. for Lyn) and anti-Fyn tended not to stain small B-cell neoplasms. The observation that a range of leucocyte signalling molecules can be detected in routine biopsies offers new possibilities for studying normal and neoplastic human white cells in diagnostic tissue samples. [ABSTRACT FROM AUTHOR]

The article discusses a study which appeared in the issue which examined several cases of diffuse large B-cell lymphoma (DLBCL) with a particular high content of programmed cell death-1-positive T cells. Topics include the definition of the T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) in the World Health Organization classification, clinical features of the DLBCL/THRLBCL cases, and the implications of the study findings for the differential diagnosis of peripheral T-cell lymphoma.

Hodgkin lymphoma (HL) presents with a unique histologic pattern. Pathognomonic Hodgkin and Reed–Sternberg (HRS) cells usually account for less than 1% of the tumor and are embedded in a reactive infiltrate mainly comprised of CD4+T cells. HRS cells induce an immunosuppressive microenvironment and thereby escape antitumor immunity. To investigate the impact of interactions between HRS cells and T cells, we performed long-term co-culture studies that were further translated into a xenograft model. Surprisingly, we revealed a strong antitumor potential of allogeneic CD4+T cells against HL cell lines. HRS and CD4+T cells interact by adhesion complexes similar to immunological synapses. Tumor-cell killing was likely based on the recognition of allogeneic major histocompatibility complex class II (MHC-II) receptor, while CD4+T cells from MHC-II compatible donors did not develop any antitumor potential in case of HL cell line L428. However, gene expression profiling (GEP) of co-cultured HRS cells as well as tumor infiltration of matched CD4+T cells indicated cellular interactions. Moreover, matched CD4+T cells could be activated to kill CD30+HRS cells when redirected with a CD30-specific chimeric antigen receptor. Our work gives novel insights into the crosstalk between HRS and CD4+T cells, suggesting the latter as potent effector cells in the adoptive cell therapy of HL. [ABSTRACT FROM AUTHOR]

Germinal centers (GCs) are some of the most important structures in the human immune system. As such, their cell types and functions have been thoroughly investigated. B cells, T cells, follicular dendritic cells (FDCs), and macrophages have widely been found to typically be aggregated in GCs. However, the amount of space occupied by each of these cell types has yet to be investigated. In this study, we conducted confocal laser-based 3D cell-volume quantification of typical GC cells under reactive conditions in lymphadenitis and investigated how volume proportions change during GC development. For this investigation, we used anti-CD3 (T cells), anti-CD20 and anti-Pax5 (B cells), anti-CD23 (FDCs), anti-CD68 (macrophages), and DAPI (nuclear staining). We detected average proportions of about 11% CD3, 9% CD20, 6% CD23, and 2% CD68 in the largest possible regions of interest within GCs. Interestingly, these values remained steady relatively independent of GC size. The remarkably low B cell proportion can be attributed to technical constraints given the use of the CD20 antibody in 3D. Applying the B cell marker Pax5, we found that about 44% of the volume was occupied by B cells after extrapolating the volume of B cell nuclei to that of whole B cells. We concluded that Pax5 is more suitable than anti-CD20 for 3D B cell quantification in GCs. The substantial unstained volume in GCs raises the question of whether other cell types fill these open spaces. Our 3D investigation enabled a unique morphological and volumetric evaluation of GC cells that balance their overall volumes in GCs. [ABSTRACT FROM AUTHOR]