Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior.

Burkitt lymphoma (BL) is characterized by tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to a favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of EBV + BL with granulomatous reaction compared to 8 cases of EBV + BL and 8 EBV- BL, both with typical "starry sky" pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated by multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (Cluster 2), EBV - BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky", both EBV + and EBV -. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, while BLs with "starry sky" were characterized by up-regulation of M2- polarization and pro-tumor response. Moreover, the analysis of additional signatures revealed an up-regulation of Dark zone-signature and epigenetic-signature in BL with typical "starry sky". Tumor associated macrophages (TAM) and epigenetic regulators may be promising targets for additional therapies in BL lymphoma opening novel immunotherapeutic strategies.

A Microenvironment-Related Nine-Gene Signature May Predict Survival in Mycosis Fungoides Patients at Diagnosis.

Mycosis fungoides (MF) is the most common cutaneous lymphoma characterized by an indolent course. Prognosis is stage-based but this approach does not reflect the different outcomes within stages. Considering that tumor microenvironment is known to be involved in MF pathogenesis and progression, we decided to investigate 99 MF cases by using the PanCancer Immune Profiling Panel. We identified and validated a signature of 9 genes able to predict MF survival and distinguish a high-risk group with a worse outcome from a low-risk group of cases with a better outcome. At the molecular level, low-risk vs. high-risk cases reported a global upregulation of immune genes, enriched in cytokines, and a higher density of dendritic cells and mast cells, possibly associated with a more favorable clinical course.

NR1H3 (LXRα) is associated with pro-inflammatory macrophages, predicts survival and suggests potential therapeutic rationales in diffuse large b-cell lymphoma.

The role of macrophages (Mo) and their prognostic impact in diffuse large B-cell lymphomas (DLBCL) remain controversial. By regulating the lipid metabolism, Liver-X-Receptors (LXRs) control Mo polarization/inflammatory response, and their pharmacological modulation is under clinical investigation to treat human cancers, including lymphomas. Herein, we surveyed the role of LXRs in DLBCL for prognostic purposes. Comparing bulk tumors with purified malignant and normal B-cells, we found an intriguing association of NR1H3, encoding for the LXR-α isoform, with the tumor microenvironment (TME). CIBERSORTx-based purification on large DLBCL datasets revealed a high expression of the receptor transcript in M1-like pro-inflammatory Mo. By determining an expression cut-off of NR1H3, we used digital measurement to validate its prognostic capacity on two large independent on-trial and real-world cohorts. Independently of classical prognosticators, NR1H3high patients displayed longer survival compared with NR1H3low cases and a high-resolution Mo GEP dissection suggested a remarkable transcriptional divergence between subgroups. Overall, our findings indicate NR1H3 as a Mo-related biomarker identifying patients at higher risk and prompt future preclinical studies investigating its mouldability for therapeutic purposes.

The identification of TCF1+ progenitor exhausted T cells in THRLBCL may predict a better response to PD-1/PD-L1 blockade.

T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a rare and aggressive variant of diffuse large B-cell lymphoma (DLBCL) that usually affects young to middle-aged patients, with disseminated disease at presentation. The tumor microenvironment (TME) plays a key role in THRLBCL due to its peculiar cellular composition (<10% neoplastic B cells interspersed in a cytotoxic T-cell/histiocyte-rich background). A significant percentage of THRLBCL is refractory to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP)-based regimens and to chimeric antigen receptor T-cell therapy; thus, the development of a specific therapeutic approach for these patients represents an unmet clinical need. To better understand the interaction of immune cells in THRLBCL TME and identify more promising therapeutic strategies, we compared the immune gene expression profiles of 12 THRLBCL and 10 DLBCL samples, and further corroborated our findings in an extended in silico set. Gene coexpression network analysis identified the predominant role of the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis in the modulation of the immune response. Furthermore, the PD-1/PD-L1 activation was flanked by the overexpression of 48 genes related to the functional exhaustion of T cells. Globally, THRLBCL TME was highly interferon-inflamed and severely exhausted. The immune gene profiling findings strongly suggest that THRLBCL may be responsive to anti-PD-1 therapy but also allowed us to take a step forward in understanding THRLBCL TME. Of therapeutic relevance, we validated our results by immunohistochemistry, identifying a subset of TCF1+ (T cell-specific transcription factor 1, encoded by the TCF7 gene) progenitor exhausted T cells enriched in patients with THRLBCL. This subset of TCF1+ exhausted T cells correlates with good clinical response to immune checkpoint therapy and may improve prediction of anti-PD-1 response in patients with THRLBCL.

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Molecular Features of Blastic Plasmacytoid Dendritic Cell Neoplasm: DNA Mutations and Epigenetics.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic neoplasm with a dismal prognosis and no standard therapy. In the past, its cellular ontogenesis was obscure, and BPDCN had been erroneously named CD56+/TdT+ blastic NK cell tumor and CD4+/CD56+ hematodermic neoplasm. Finally, in 2008, the BPDCN was correctly recognized as a neoplasm deriving from the malignant transformation of plasmacytoid dendritic cell precursors and classified among the myeloid neoplasms. Since then, the understanding of BPDCN biology has improved rapidly: the DNA mutational status of BPDCN has been extensively investigated revealing a spectrum perfectly resembling its myeloid lineage derivation.

Correction: Whole exome sequencing reveals mutations in FAT1 tumor suppressor gene clinically impacting on peripheral T-cell lymphoma not otherwise specified.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole exome sequencing reveals mutations in FAT1 tumor suppressor gene clinically impacting on peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphoma not otherwise specified represents a diagnostic category comprising clinically, histologically, and molecularly heterogeneous neoplasms that are poorly understood. The genetic landscape of peripheral T-cell lymphoma not otherwise specified remains largely undefined, only a few sequencing studies having been conducted so far. In order to improve our understanding of the genetics of this neoplasm, we performed whole exome sequencing along with RNA-sequencing in a discovery set of 21 cases. According to whole exome sequencing results and mutations previously reported in other peripheral T-cell lymphomas, 137 genes were sequenced by a targeted deep approach in 71 tumor samples. In addition to epigenetic modifiers implicated in all subtypes of T-cell neoplasm (TET2, DNMT3A, KMT2D, KMT2C, SETD2), recurrent mutations of the FAT1 tumor suppressor gene were for the first time recorded in 39% of cases. Mutations of the tumor suppressor genes LATS1, STK3, ATM, TP53, and TP63 were also observed, although at a lower frequency. Patients with FAT1 mutations showed inferior overall survival compared to those with wild-type FAT1. Although peripheral T-cell lymphoma not otherwise specified remains a broad category also on molecular grounds, the present study highlights that FAT1 mutations occur in a significant proportion of cases, being provided with both pathogenetic and prognostic impact.

Blastic Plasmacytoid Dendritic Cell Neoplasm: State of the Art and Prospects.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an extremely rare tumour, which usually affects elderly males and presents in the skin with frequent involvement of the bone-marrow, peripheral blood and lymph nodes. It has a dismal prognosis, with most patients dying within one year when treated by conventional chemotherapies. The diagnosis is challenging, since neoplastic cells can resemble lymphoblasts or small immunoblasts, and require the use of a large panel of antibodies, including those against CD4, CD56, CD123, CD303, TCL1, and TCF4. The morphologic and in part phenotypic ambiguity explains the uncertainties as to the histogenesis of the neoplasm that led to the use of various denominations. Recently, a series of molecular studies based on karyotyping, gene expression profiling, and next generation sequencing, have largely unveiled the pathobiology of the tumour and proposed the potentially beneficial use of new drugs. The latter include SL-401, anti-CD123 immunotherapies, venetoclax, BET-inhibitors, and demethylating agents. The epidemiologic, clinical, diagnostic, molecular, and therapeutic features of BPDCN are thoroughly revised in order to contribute to an up-to-date approach to this tumour that has remained an orphan disease for too long.

Blastic plasmacytoid dendritic cell neoplasm: genomics mark epigenetic dysregulation as a primary therapeutic target.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy for which there is still no effective therapy. In order to identify genetic alterations useful for a new treatment design, we used whole-exome sequencing to analyze 14 BPDCN patients and the patient-derived CAL-1 cell line. The functional enrichment analysis of mutational data reported the epigenetic regulatory program to be the most significantly undermined (P<0.0001). In particular, twenty-five epigenetic modifiers were found mutated (e.g. ASXL1, TET2, SUZ12, ARID1A, PHF2, CHD8); ASXL1 was the most frequently affected (28.6% of cases). To evaluate the impact of the identified epigenetic mutations at the gene-expression and Histone H3 lysine 27 trimethylation/acetylation levels, we performed additional RNA and pathology tissue-chromatin immunoprecipitation sequencing experiments. The patients displayed enrichment in gene signatures regulated by methylation and modifiable by decitabine administration, shared common H3K27-acetylated regions, and had a set of cell-cycle genes aberrantly up-regulated and marked by promoter acetylation. Collectively, the integration of sequencing data showed the potential of a therapy based on epigenetic agents. Through the adoption of a preclinical BPDCN mouse model, established by CAL-1 cell line xenografting, we demonstrated the efficacy of the combination of the epigenetic drugs 5'-azacytidine and decitabine in controlling disease progression in vivo.

Ruxolitinib in elderly patients with myelofibrosis: impact of age and genotype. A multicentre study on 291 elderly patients.

Ruxolitinib is a JAK1/2 inhibitor that may control myelofibrosis (MF)-related splenomegaly and symptoms and can be prescribed regardless of age. While aging is known to correlate with worse prognosis, no specific analysis is available to confirm that ruxolitinib is suitable for use in older populations. A clinical database was created in 23 European Haematology Centres and retrospective data on 291 MF patients treated with ruxolitinib when aged ≥65 years were analysed in order to assess the impact of age and molecular genotype on responses, toxicities and survival. Additional mutations were evaluated by a next generation sequencing (NGS) approach in 69 patients with available peripheral blood samples at the start of ruxolitinib treatment. Compared to older (age 65-74 years) patients, elderly (≥75 years) showed comparable responses to ruxolitinib, but higher rates of drug-induced anaemia and thrombocytopenia and worse survival. Nonetheless, the ruxolitinib discontinuation rate was comparable in the two age groups. Number and types of molecular abnormalities were comparable across age groups. However, the presence of high molecular risk (HMR) mutations significantly affected survival, counterbalancing the effect of aging. Indeed, elderly patients with <2 HMR mutated genes had a comparable survival to older patients with ≥2 HMR mutations. Given that responses were not influenced by age, older age per se should not be a limitation for ruxolitinib administration. NGS analysis of HMR mutations also confirmed a strong predictive value in elderly patients.

Distinctive Histogenesis and Immunological Microenvironment Based on Transcriptional Profiles of Follicular Dendritic Cell Sarcomas.

Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumors with variable clinical, morphologic, and phenotypic characteristics. Transcriptome analysis was performed on multiple FDC sarcomas and compared with other mesenchymal tumors, microdissected Castleman FDCs, and normal fibroblasts. Using unsupervised analysis, FDC sarcomas clustered with microdissected FDCs, distinct from other mesenchymal tumors and fibroblasts. The specific endowment of FDC-related gene expression programs in FDC sarcomas emerged by applying a gene signature of differentially expressed genes (n = 1,289) between microdissected FDCs and fibroblasts. Supervised analysis comparing FDC sarcomas with microdissected FDCs and other mesenchymal tumors identified 370 and 2,927 differentially expressed transcripts, respectively, and on the basis of pathway enrichment analysis ascribed to signal transduction, chromatin organization, and extracellular matrix organization programs. As the transcriptome of FDC sarcomas retained similarity with FDCs, the immune landscape of FDC sarcoma was investigated by applying the CIBERSORT algorithm to FDC sarcomas and non-FDC mesenchymal tumors and demonstrated that FDC sarcomas were enriched in T follicular helper (TFH) and T regulatory (TREG) cell populations, as confirmed in situ by immunohistochemistry. The enrichment in specific T-cell subsets prompted investigating the mRNA expression of the inhibitory immune receptor PD-1 and its ligands PD-L1 and PD-L2, which were found to be significantly upregulated in FDC sarcomas as compared with other mesenchymal tumors, a finding also confirmed in situ Here, it is demonstrated for the first time the transcriptional relationship of FDC sarcomas with nonmalignant FDCs and their distinction from other mesenchymal tumors.Implications: The current study provides evidence of a peculiar immune microenvironment associated with FDC sarcomas that may have clinical utility. Mol Cancer Res; 15(5); 541-52. ©2017 AACR.

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.

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.

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.

Glutathione transferase-A2 S112T polymorphism predicts survival, transplant-related mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation.

Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36 ± 570.06 vs. 838.10 ± 282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280 ± 0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.

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.