Frozen samples from 92 malignant astrocytomas were stained with a panel of monoclonal antibodies directed against macrophages and lymphocytes. A follow-up to death was available on 68 cases which form the basis of this study. Large numbers of macrophages were found in all cases; T lymphocytes, mostly of the CD8 phenotype were also seen in moderate numbers in 70% of cases. CD4-positive cells were present in small numbers in 32% and B cells were seen in only 8% of cases. Analysis of the survival showed no demonstrable correlation between the numbers of macrophages or CD4 lymphocytes and survival. The survival curves for parenchymal CD8 infiltration diverged after 9 months suggesting increased survival for those patients without such an infiltration but the difference failed to reach statistical significance (P = 0.37). No correlation between lymphocytic cuffing and survival was seen after studying all paraffin-embedded material. We conclude that there is no significant statistical correlation between survival and the various types of mononuclear cell infiltrating malignant astrocytomas.
773 Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous tumors whose biology is largely unknown. Interestingly, the commonest subtypes (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) present on one hand few disease-specific molecular features and, on the other hand, several apparently common abnormalities. So far, no data are available regarding miRNA expression in these tumors. In order to identify miRNA deregulated in PTCLs, we performed an extensive miRNA profiling (by studying 379 targets on the TaqMan Array MicroRNA Cards) of 44 PTCLs (including 23 PTCLs/NOS, 12 ALCLs, and 9 AITLs) and 13 sample representative of normal T-cell sub-populations (CD4+ and CD8+, both resting and activated). In addition, for all these cases, gene expression profiles (GEPs) were generated by the Ilumina whole genome DASL-assay. TaqMan Quantitative-PCR (qPCR) was then used for validation. First, we found that PTCLs and normal T-cells could be easily distinguished based on their miRNA profile, by both unsupervised and supervised analysis. Specifically, the latter identified 91 miRNA differentially expressed in PTCLs vs. T-cells with a fold change ≥2 and a pvalue<0.01. Interestingly, the predicted target genes of such miRNA were involved in relevant cellular functions (such as cell proliferation, apoptosis, signal transduction etc.) and were found to be differentially expressed in tumor vs. controls, indicating a functional impact of miRNA deregulation in the molecular phenotype of neoplastic T-cells. Intriguingly, several drugs were estimated in silico to be able to interfere with pathways controlled by the de-regulated miRNA, including flavopiridol, everolimus/ temsirolimus, and forodesine. Secondly, by supervised analysis (ANOVA), we identified miRNA differentially expressed among PTCL/NOS, AITL and ALCL. Indeed, basing on such miRNA it was possible to roughly separate the different entities by hierarchical clustering (HC) (chi-square, p=0.0005). Specifically, we found 7 miRNA differentially expressed in PTCL/NOS vs. AITL, 26 in PTCL/NOS vs. ALK- ALCL, and 16 in ALK- vs. ALK+ ALCL. Selected miRNA were then studied by qPCR in an independent set of PTCLs (including 10 PTCL/NOS, 5 AITL, 5 ALK- ALCL, and 5 ALK+ ALCL) as for validation. Subsequently, in order to assess the impact of miRNA on the GEP, we picked up the miRNA most efficient in differentiating the diverse tumors (miR-431, miR-887, miR-889, and miR-155), and we studied the expression of their predicted target genes. Indeed, we found that miR-431 target genes were differentially expressed in PTCL/NOS vs. AITL (GSEA, q-value=0.45), and basing on their expression, HC could efficiently divide the two diseases (chi-square, p=0.004). Similarly, miR-889 targets were differentially expressed in PTCL/NOS vs. ALK- ALCL (GSEA, q-value=0; chi-square for HC, p=0.01), while miR-155 targets were differentially expressed in ALK+ vs. ALK- ALCL (GSEA, q-value=0; chi-square for HC, p<0.001). In conclusion, we performed for the first time an extensive miRNA profiling of PTCLs and combined it with global GEP. Importantly we identified miRNAs deregulated in PTCLs with possible pathogenetic implications. In addition, we found miRNAs, whose detection might be relevant for the differential diagnosis of PTCL subtypes. Finally, we provided in silico evidences that the cellular pathway controlled by de-regulated miRNA in PTCLs might serve as potential therapeutic targets, thus warranting further functional investigation. Disclosures: No relevant conflicts of interest to declare.
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. In this study, we aimed to: 1) molecularly define the cellular counterpart of BPDCN and its relationship with other leukemias; 2) identify genes and cellular programs deregulated in the tumor; and 3) delineate novel potential therapeutic targets. To address these issues we collected and studied by gene expression profile (GEP) 27 BPDCN cases as well as 8 samples of non neoplastic pDCs. Further, a panel of samples including, myeloid precursors (MPs, N=4), lymphoid precursors (LPs, N=9), acute myeloid leukemias (AMLs, N=132), and acute lymphoblastic leukemias (ALLs, N=155) was analyzed. Validation was performed by immunohistochemistry (IHC) on tissue-microarrays, while functional experiments were carried out by using the CAL-1 cell line (derived from a BPDCN case). First, we recognized the cellular derivation of BPDCN, which proved to originate from the myeloid lineage and in particular from resting pDCs. Second, by comparing the GEP of BPDCN and resting pDCs, we identified genes and cellular programs deregulated in the tumor. Following, based on an integrated bio-informatic approach, including four different tools, we uncovered the aberrant activation of the NF-kB pathway that was confirmed in independent assays. Interestingly, among other molecules, we identified BCL2 and IRF4, two well known NFkB targets, as aberrantly upregulated in neoplastic samples and confirmed this observation by IHC. We then tested whether NFkB inhibition could represent a potential therapeutic strategy in this setting. We treated BPDCN cells ex vivowith either the proteasome inhibitor bortezomib or the selective IKKB inhibitor BMS-345541 and found them to be effective in inducing cell cycle arrest and apoptosis at relatively low dosage. By contrast, BPDCN cells turned out to be virtually insensitive to cytarabine, one of the most used drug in this condition. GEP and immunocytochemistry were then successfully used to prove that cell death was accompanied by NFkB shut-off. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing the first molecular targeted therapeutic approach in the setting of this currently incurable disease. Funding This work was supported by AIRC (IG10519 and 5xMille10007, Prof. Pileri), Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, RFO (Prof. Pileri, Prof. Piccaluga), FIRB Futura 2011 RBFR12D1CB (Prof. Piccaluga), Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006 (Prof. Pileri and Dr. Piccaluga) and by MIUR (PRIN 2011, Prof. Facchetti and Prof. Pileri). The authors have no conflicting financial interests to declare. Acknowledgments The Authors obtained the CAL-1 cell line from Takahiro Maeda (tmaeda@net.nagasaki-u.ac.jp), Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. Disclosures: No relevant conflicts of interest to declare.
3662 The differential diagnosis among the commonest peripheral T-cell lymphomas (PTCLs) (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) is difficult, the morphologic and phenotypic features being largely overlapping. Noteworthy, recent international studies indicated significant differences in their clinical behavior as well as concerning the presence of potential therapeutic targets. We performed whole genome gene expression profiling (GEP) of PTCLs aiming to identify molecular signatures able to improve their diagnosis. We studied 95 PTCLs, including 73 PTCLs/NOS, 12 ALCLs (6 ALK+ and 6 ALK-), and 10 AITLs. All tissue samples were formalin-fixed and paraffin embedded (FFPE). GEP was performed by Illumina Whole Genome DASL Assay. First, we documented the efficiency of GEP from FFPE tissues by comparing the mRNA levels and the presence of the corresponding protein, including expressed (i.e. CD3) and not expressed (i.e. BCL10) molecules. Secondly, we tried to discriminate different PTCLs basing on their GEPs. By dividing a training (N=47) and a test set (N=48), we found 2 signatures able to differentiate PTCL/NOS vs. AITL and PTCL/NOS vs. ALCL ALK-. Specifically, in the test set the sensitivity (ST) and specificity (SP) of the assays were 100% – 80% (PTCL/NOS vs. AITL) and 100% – 100% (PTCL/NOS vs. ALK- ALCL) (Table 1). Accordingly, the positive (PPV) and negative (NPV) predicting values for the identification of PTCL/NOS were 0.92 and 1 (vs. AITL) and 1 and 1 (vs. ALK- ALCL) (Table 1).Table 1.Accuracy of GEP based signature in differentiating PTCL subtypesSTSPPPVNPVTraining setPTCL/NOS vs. AITL100%80%0.921PTCL/NOS vs. ALK-ALCL100%100%11Test setPTCL/NOS vs. AITL92.50%100%10.77PTCL/NOS vs. ALK-ALCL92.50%100%10.67Validation setPTCL/NOS vs. AITL85%86%0.920.76PTCL/NOS vs. ALK-ALCL96%73%0.960.73 Interestingly, the identified genes represented relevant functional pathways differentially regulated in the 3 tumour types, including protein kinase cascade, proliferation, and cell cycle. When applied to the test set of cases, the assay correctly classified 37/40 PTCLs/NOS (92.5%), 5/5 AITLs, and 3/3 ALK- ALCLs. Finally, we tested our signatures on 133 independent PTCL cases (including 78 PTCL/NOS, 43 AITL, and 12 ALK- ALCL) for which GEP data were available on the GEO database and were originally obtained from fresh/frozen tissues. Interestingly, we could efficiently recognize PTCL/NOS cases vs. AITLs (ST, 85%; SP 86%; PPV 0.92; NPV 0.76) and vs. ALK- ALCLs (ST 96%; SP 73%; PPV 0.96; NPV 0.73). In conclusion, we successfully generated for the first time GEP from routinary FFPE PTCL samples, identifying molecular signatures potentially useful for the clinical practice and, specifically, for the differential diagnosis of PTCL types. Disclosures: No relevant conflicts of interest to declare.
Tumors derived from histiocytes and follicular dendritic cells (FDC) are extremely rare, constituting less than 1% of hematopoietic tumors. Among others, follicular dendritic cell sarcoma (FDC-S), histiocytic sarcoma (HS) , and Langerhans histiocytosis (LH) are the commonest types. The clinical course is variable from indolent to aggressive; however, the disease is more often incurable. Further, probably due to their rarity and complexity, the pathobiology of these tumor is nearly unknown at present, with only scattered studies being reported. In this study, we investigated by gene expression profiling (GEP) and extensive immunohistochemistry (IHC) a relatively large series of cases, aiming 1) to identify and characterize functional pathways possibly involved in the pathogenesis of the disease, and 2) to unveil potential targets for novel treatments. We studied by GEP FDC-S (N=30), LH (N=9), and HS (N=4) as well as non neoplastic cells including tissue microdissected FDC (N=5), CD1c+ myeloid dendritic cells (N=4), peripheral blood monocytes (N=8), and human cultured fibroblasts (N=3). All cases were studied with the Illumina DASL whole genome microarray. The same cases as well as and independent cohort was then used for IHC validation studies. First, unsupervised approaches (principal component analysis and hierarchical clustering) indicated that tissue samples were distinct from cellular ones, while the different entities could not be clearly separated. Second, we found by supervised comparison (ANOVA, p<0.05, fold change ≥2, FDR according to Benjamini-Hockeberg), the 3 tumor types could be discriminated. Particularly, the tumors differed for genes related to immune response (consistent with their histogenesis) and to intracellular signaling. In this regard, we found several oncogenic pathways to be differentially activated while others were consistently deregulated including those related to cellular adhesion, chemotaxis, angiogenesis and response to growth factors. We then compared each tumor type with the specific supposed counterpart (i.e. FDC-s vs. FDC; LH vs. myeloid DC; HS vs. monocytes) and identified genes and cellular programs deregulated in each of them. Interestingly, all tumors presented with consistent deregulation of previously unknown potential therapeutic targets, including PDGF/PDGFRs, PTEN, JAK/STAT, mTOR/AKT, and EGFR. Importantly, IHC confirmed GEP data and confirmed the activation of these selected pathways. We defined for the first time the cellular programs deregulated in tumors derived from histiocytes and FDC and identified remarkable potential therapeutic targets. As appropriate experimental models (i.e. cell lines, animal models) are not available at present, international collaboration would be advisable to confirm these observation in large series before eventually moving to early phase clinical trials. Citation Format: Pier-Paolo Piccaluga, Maura Rossi, Giovanna Motta, Sylvia Hartmann, Claudia Doering, Fabio Fuligni, Claudio Agostinelli, Maria Rosaria Sapienza, Maria Antonella Laginestra, Federica Melle, Maryam Etebari, Mohsen Navari, Anna Gazzola, Claudia Mannu, Clara Bertuzzi, Claudio Tripodo, Martin L. Hansmann, Fabio Facchetti, Stefano A Pileri. Identification and characterization of pathogenetic pathways and potential therapeutic targets in tumors derived from histiocytes and follicular dendritic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2014-2362
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