Fabio Fuligni

Molecular Profiling Improves Classification and Prognostication of Nodal Peripheral T-Cell Lymphomas: Results of a Phase III Diagnostic Accuracy Study

PURPOSE The differential diagnosis among the commonest peripheral T-cell lymphomas (PTCLs; ie, PTCL not otherwise specified [NOS], angioimmunoblastic T-cell lymphoma [AITL], and anaplastic large-cell lymphoma [ALCL]) is difficult, with the morphologic and phenotypic features largely overlapping. We performed a phase III diagnostic accuracy study to test the ability of gene expression profiles (GEPs; index test) to identify PTCL subtype. METHODS We studied 244 PTCLs, including 158 PTCLs NOS, 63 AITLs, and 23 ALK-negative ALCLs. The GEP-based classification method was established on a support vector machine algorithm, and the reference standard was an expert pathologic diagnosis according to WHO classification. RESULTS First, we identified molecular signatures (molecular classifier [MC]) discriminating either AITL and ALK-negative ALCL from PTCL NOS in a training set. Of note, the MC was developed in formalin-fixed paraffin-embedded (FFPE) samples and validated in both FFPE and frozen tissues. Second, we found that the overall accuracy of the MC was remarkable: 98% to 77% for AITL and 98% to 93% for ALK-negative ALCL in test and validation sets of patient cases, respectively. Furthermore, we found that the MC significantly improved the prognostic stratification of patients with PTCL. Particularly, it enhanced the distinction of ALK-negative ALCL from PTCL NOS, especially from some CD30+ PTCL NOS with uncertain morphology. Finally, MC discriminated some T-follicular helper (Tfh) PTCL NOS from AITL, providing further evidence that a group of PTCLs NOS shares a Tfh derivation with but is distinct from AITL. CONCLUSION Our findings support the usage of an MC as additional tool in the diagnostic workup of nodal PTCL.

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.

Defective Stromal Remodeling and Neutrophil Extracellular Traps in Lymphoid Tissues Favor the Transition from Autoimmunity to Lymphoma

Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

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.

Bone marrow stroma CD40 expression correlates with inflammatory mast cell infiltration and disease progression in splenic marginal zone lymphoma

Splenic marginal zone lymphoma (SMZL) is a mature B-cell neoplasm characterized by rather indolent clinical course. However, nearly one third of patients experience a rapidly progressive disease with a dismal outcome. Despite the characterization of clone genetics and the recognition of deregulated immunologic stimulation in the pathogenesis of SMZL, little is known about microenvironment dynamics and their potential biological influence on disease outcome. Here we investigate the effect of stroma-intrinsic features on SMZL disease progression by focusing on the microenvironment of the bone marrow (BM), which represents an elective disease localization endorsing diagnostic and prognostic relevance. We show that the quality of the BM stromal meshwork of SMZL infiltrates correlates with time to progression. In particular, we describe the unfavorable prognostic influence of dense CD40 expression by BM stromal cells, which involves the contribution of CD40 ligand (CD40L)-expressing bystander mast cells infiltrating SMZL BM aggregates. The CD40/CD40L-assisted crosstalk between mesenchymal stromal cells and mast cells populating the SMZL microenvironment finds correlation in p53(-/-) mice developing SMZL and contributes to the engendering of detrimental proinflammatory conditions. Our study highlights a dynamic interaction, playing between nonneoplastic elements within the SMZL niche, toward disease progression.

Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas.

Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown. In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs. First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones. In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA.

Molecular signature of Epstein Barr virus-positive Burkitt lymphoma and post-transplant lymphoproliferative disorder suggest different roles for Epstein Barr virus.

Epstein Barr virus (EBV) infection is commonly associated with human cancer and, in particular, with lymphoid malignancies. Although the precise role of the virus in the pathogenesis of different lymphomas is largely unknown, it is well recognized that the expression of viral latent proteins and miRNA can contribute to its pathogenetic role. In this study, we compared the gene and miRNA expression profile of two EBV-associated aggressive B non-Hodgkin lymphomas known to be characterized by differential expression of the viral latent proteins aiming to dissect the possible different contribution of such proteins and EBV-encoded miRNAs. By applying extensive bioinformatic inferring and an experimental model, we found that EBV+ Burkitt lymphoma presented with significant over-expression of EBV-encoded miRNAs that were likely to contribute to its global molecular profile. On the other hand, EBV+ post-transplant diffuse large B-cell lymphomas presented a significant enrichment in genes regulated by the viral latent proteins. Based on these different viral and cellular gene expression patterns, a clear distinction between EBV+ Burkitt lymphoma and post-transplant diffuse large B-cell lymphomas was made. In this regard, the different viral and cellular expression patterns seemed to depend on each other, at least partially, and the latency type most probably played a significant role in their regulation. In conclusion, our data indicate that EBV influence over B-cell malignant clones may act through different mechanisms of transcriptional regulation and suggest that potentially different pathogenetic mechanisms may depend upon the conditions of the interaction between EBV and the host that finally determine the latency pattern.

The combination of hypomethylating agents and histone deacetylase inhibitors produce marked synergy in preclinical models of T-cell lymphoma

T‐cell lymphomas (TCL) are aggressive lymphomas usually treated with CHOP (cyclophsophamide, doxorubicin, vincristine, prednisolone)‐like regimens upfront. Recent data suggest that TCL are driven by epigenetic defects, potentially rendering them sensitive to epigenetic therapies. We explored the therapeutic merits of a combined epigenetic platform using histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMT) in in vitro and in vivo models of TCL. The 50% inhibitory concentration (IC50) values revealed romidepsin was the most potent HDACI, with an IC50 in the low nanomolar range. The combination with a hypomethylating agent produced synergy across all cell lines, which was confirmed in cytotoxicity and apoptosis assays. An in vivo xenograft study demonstrated inhibition of tumour growth in the combination cohort compared to the single agent. Gene expression array and global methylation profiling revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. Most of the effects induced by the single agent treatment were maintained in the combination group. In total, 944 unique genes were modulated by the combination treatment, supporting the hypothesis of molecular synergism. These data suggest combinations of hypomethylating agents and HDACIs are synergistic in models of TCL, which is supported at the molecular level.

The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies

Currently, distinguishing between benign and malignant lymphoid proliferations is based on a combination of clinical characteristics, cyto/histomorphology, immunophenotype and the identification of well-defined chromosomal aberrations. However, such diagnoses remain challenging in 10–15% of cases of lymphoproliferative disorders, and clonality assessments are often required to confirm diagnostic suspicions. In recent years, the development of new techniques for clonality detection has allowed researchers to better characterize, classify and monitor hematological neoplasms. In the past, clonality was primarily studied by performing Southern blotting analyses to characterize rearrangements in segments of the IG and TCR genes. Currently, the most commonly used method in the clinical molecular diagnostic laboratory is polymerase chain reaction (PCR), which is an extremely sensitive technique for detecting nucleic acids. This technique is rapid, accurate, specific, and sensitive, and it can be used to analyze small biopsies as well as formalin-fixed paraffin-embedded samples. These advantages make PCR-based approaches the current gold standard for IG/TCR clonality testing. Since the completion of the first human genome sequence, there has been a rapid development of technologies to facilitate high-throughput sequencing of DNA. These techniques have been applied to the deep characterization and classification of various diseases, patient stratification, and the monitoring of minimal residual disease. Furthermore, these novel approaches have the potential to significantly improve the sensitivity and cost of clonality assays and post-treatment monitoring of B- and T-cell malignancies. However, more studies will be required to demonstrate the utility, sensitivity, and benefits of these methods in order to warrant their adoption into clinical practice. In this review, recent developments in clonality testing are examined with an emphasis on highly sensitive systems for improving diagnostic workups and minimal residual disease assessments.