Simona Righi

Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4(+), CD8(+), and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4(+), rather than to resting or CD8(+) lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-kappaB (NF-kappaB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor alpha) and VEGF.

Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.

Peripheral T cell lymphoma, not otherwise specified: the stuff of genes, dreams and therapies

Peripheral T cell lymphomas (PTCL) account for about 12% of lymphoid tumours worldwide. Almost half show such morphological and molecular variability as to hamper any further classification, and to justify their inclusion in a waste-basket category termed “not otherwise specified (NOS)”. The latter term is used for neoplasms with aggressive presentation, poor response to therapy and dismal prognosis. In contrast to B cell lymphomas, PTCL have been the subject of only a limited number of studies to elucidate their pathobiology and identify novel pharmacological approaches. Herewith, the authors revise the most recent contributions on the subject based on the experience they have gained in the extensive application of microarray technologies. PTCL/NOS are characterised by erratic expression of T cell associated antigens, including CD4 and CD52, which have recently been proposed as targets for ad hoc immunotherapies. PTCL/NOS also show variable Ki-67 marking, with rates >80% heralding a worse prognosis. Gene expression profiling studies have revealed that PTCL/NOS derive from activated T lymphocytes, more often of the CD4+ type, and bear a signature composed of 155 genes and related products that play a pivotal role in cell signalling transduction, proliferation, apoptosis and matrix remodelling. This observation seems to pave the way for the use of innovative drugs such as tyrosine kinase and histone deacetylase inhibitors whose efficacy has been proven in PTCL primary cell cultures. Gene expression profiling also allows better distinction of PTCL/NOS from angioimmunoblastic T cell lymphoma, the latter being characterised by follicular T helper lymphocyte derivation and CXCL13, PD1 and vascular endothelial growth factor expression.

Peripheral T cell lymphomas with follicular T helper phenotype: a new basket or a distinct entity? Revising Karl Lennert’s personal archive

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) Histopathology59, 679–691

Proliferation centers in chronic lymphocytic leukemia: correlation with cytogenetic and clinicobiological features in consecutive patients analyzed on tissue microarrays.

To better define the significance of proliferation centers (PCs), the morphological hallmark of chronic lymphocytic leukemia (CLL), lymph node biopsies taken from 183 patients were submitted to histopathologic and fluorescence in situ hybridization (FISH) studies using a 5-probe panel on tissue microarrays. Seventy-five cases (40.9%) with confluent PCs were classified as ‘PCs-rich’ and 108 cases (59.1%) with scattered PCs were classified as ‘typical’. Complete FISH data were obtained in 101 cases (55.1%), 79 of which (78.2%) displayed at least one chromosomal aberration. The incidence of each aberration was: 13q- 36,7%, 14q32 translocations 30.8%, 11q- 24.7%, trisomy 12 19.5% and 17p- 15.6%. Five cases showed extra copies of the 14q32 region. The ‘PCs-rich’ group was associated with 17p-, 14q32/IgH translocation, +12, Ki-67>30%. The median survival from the time of tissue biopsy for PCs-rich and typical groups was 11 and 64 months, respectively (P=0.00001). The PCs-rich pattern was the only predictive factor of an inferior survival at multivariate analysis (P=0.022). These findings establish an association between cytogenetic profile and the amount of PC in CLL, and show that this histopathologic characteristic is of value for risk assessment in patients with clinically significant adenopathy.

Pathobiology of hodgkin lymphoma.

Despite its well-known histological and clinical features, Hodgkins lymphoma (HL) has recently been the object of intense research activity, leading to a better understanding of its phenotype, molecular characteristics, histogenesis, and possible mechanisms of lymphomagenesis. There is complete consensus on the B-cell derivation of the tumor in most cases, and on the relevance of Epstein-Barr virus infection and defective cytokinesis in at least a proportion of patients. The REAL/WHO classification recognizes a basic distinction between lymphocyte predominance HL (LP-HL) and classic HL (cHL), reflecting the differences in clinical presentation and behavior, morphology, phenotype, and molecular features. cHL has been classified into four subtypes: lymphocyte rich, nodular sclerosing, with mixed cellularity, and lymphocyte depleted. The borders between cHL and anaplastic large-cell lymphoma have become sharper, whereas those between LP-HL and T-cell-rich B-cell lymphoma remain ill defined. Treatments adjusted to the pathobiological characteristics of the tumor in at-risk patients have been proposed and are on the way to being applied.

Rare lymphoid neoplasms coexpressing B- and T-cell antigens. The role of PAX-5 gene methylation in their pathogenesis

We report 3 cases of lymphoid neoplasms with mixed lineage features of T-, NK-, or B-cell marker expression and clonal gene rearrangement for both T-cell receptor and immunoglobulin light chain IgK. A characteristic of our cases was the lack of expression of the specific B-cell transcription factor, Pax5, which is essential for maintaining the identity and function of mature B cells during late B lymphopoiesis. In the absence of Pax5, B cells in vitro can differentiate into macrophages, dendritic cells, granulocytes, and T/NK cells. Methylation analysis of the Pax5 gene in our cases suggests that its inactivation by this epigenetic event in a committed or mature B cell, before plasma cell differentiation, may well be a common pathogenetic mechanism in mature lymphoid neoplasms with expression of multilineage antigens. In particular, case 1 may represent a mixed NK- and B-cell lineage; and cases 2 and 3 may represent mixed T and B-cell lineage, respectively. Aberrations in the DNA methylation patterns are currently recognized as a hallmark of human cancer. Cases with aberrant phenotypes require molecular analysis for lineage assignment. Studies of such cases may be helpful to better elucidate whether they represent a distinct entity with clinical, immunophenotypic, and molecular characteristics or an incidental phenomenon during malignant transformation. Interestingly, these cases were all characterized by poor clinical outcome.

Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients’ plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Detection of LIM domain only 2 (LMO2) in normal human tissues and haematopoietic and non-haematopoietic tumours using a newly developed rabbit monoclonal antibody

Agostinelli C, Paterson J C, Gupta R, Righi S, Sandri F, Piccaluga P P, Bacci F, Sabattini E, Pileri S A & Marafioti T 
(2012) Histopathology 61, 33–46

Characterization of a new monoclonal antibody against PAX5/BASP in 1525 paraffin-embedded human and animal tissue samples.

IntroductionWe describe the newly generated DAK-PAX5 monoclonal antibody raised against a fixation-resistant epitope of the human PAX5/BSAP molecule. Materials and MethodsFollowing Western-blot, absorption, and chess-board titration tests, and optimization of antigen-retrieval and detection methods, DAK-Pax5 was used in parallel with a reference antibody (clone 24) on tissue micro-arrays (TMAs) constructed from normal human and animal tissues and from hematologic and nonhematologic human malignancies. Such TMAs were also tested with an anti-PAX2 antibody. ResultsDAK-Pax5 reacted with normal human and animal B-cells and with 460/473 B-cell non-Hodgkin lymphomas (B-NHLs). All plasmacytomas/plasmablastic tumors (n=13) and T/NK-cell neoplasms (n=264) turned out consistently negative as did acute myelogenous leukaemias (n=19) except 2 carrying t(8;21). Positivity was found in 6/6 and 155/169 lymphocyte predominant and classical HLs, respectively, although the staining intensity varied through cases. Among 521 nonhematologic malignancies, DAK-Pax5 reacted with 22/399 carcinomas (4/11 neuroendocrine, 2/4 Merkel-cell, 4/21 prostatic, 1/11 urothelial, 1/26 renal, 2/12 cervical squamous-cell, 3/13 ovarian, and 5/75 colonic). When compared with clone 24, DAK-Pax5 produced a stronger positivity in most if not all B-NHLs and HLs. No cross-reactivity with the anti-PAX2 antibody was recorded. DiscussionDAK-Pax5 represents a new reliable tool for diagnostics and research.