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Enrichment analysis report: enrichment by process networks. (PDF 35 kb)

Basal expression of Chk1 and Chk2 (mRNA level) in the cell lines treated, the results are expressed as 2exp(-ΔΔCt) (A). Schematic representation of the statistically significance of the ANOVA multiple comparison test in which the basal expression of each cell lines is compared to the value of the basal expression of all the other cell lines (B). Figure S2. Oncomine expression analysis of Chk1 (A) and Chk2 (B) levels between normal monunuclear cells (MNCs) from bone marrow and different subtypes of leukemia (AML, B-ALL, T-ALL). Figure S3. Apoptosis following treatment with PF-00477736 at 24 and 48 hours in B-ALL and T-ALL cells(A). Cell cycle analysis of RPMI-8402, BV-173, SUP-B15 and NALM-6 cell lines treated for 24 hours with increasing concentrations of PF-00477736 (B). Figure S4. Western Blot analysis in all leukemia cell lines after exposure to PF-00477736 (+) at the concentration closest the IC50 or DMSO 0.1 %(-) (A). Western Blot analysis of BV-173 and NALM-6 cell lines treated with or without PF-00477736 (IC50) for 3, 6 and 9 hours (B). Figure S5. Cell cycle_ESR1 regulation of G1/S transition: the top scored map (map with the lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S6. Apoptosis and survival_Granzyme A signaling: the second scored map (map with the second lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S7. DNA damage_ATM/ATR regulation of G1/S checkpoint: the third scored map (map with the third lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S8. Protein expression level of c-Jun in B-/T-ALL cell lines (A). Western Blot analysis of normal MNCs and ALL MNCs after exposure to PF-0047773(B). mRNA expression of CDK4, Chk2, GADD45a and PLK3 in B-/T-ALL cell lines treated with or without PF-00477736(IC50 value) for 24 hours. The results are expressed as 2exp(-ΔΔCt)(C). (PPTX 2905 kb)

Table S1. List of differentially expressed genes between treated cells and their untreated counterpart with a false discovery rate less than 0.05. Table S2. Main clinical and molecular characteristics of ALL patients whose primary ALL samples have been tested in vitro. Table S3. TP53 primer sequences. Table S4. Antibody reactivity and sources as well as the antigen retrieval protocols, dilutions, and revelation systems used in immunohistochemistry. (DOCX 28 kb)

Treatment resulted in a differential expression of 941 genes ( pâ

Enrichment analysis report: enrichment by GO processes. (PDF 41 kb)

Basal expression of Chk1 and Chk2 (mRNA level) in the cell lines treated, the results are expressed as 2exp(-ΔΔCt) (A). Schematic representation of the statistically significance of the ANOVA multiple comparison test in which the basal expression of each cell lines is compared to the value of the basal expression of all the other cell lines (B). Figure S2. Oncomine expression analysis of Chk1 (A) and Chk2 (B) levels between normal monunuclear cells (MNCs) from bone marrow and different subtypes of leukemia (AML, B-ALL, T-ALL). Figure S3. Apoptosis following treatment with PF-00477736 at 24 and 48 hours in B-ALL and T-ALL cells(A). Cell cycle analysis of RPMI-8402, BV-173, SUP-B15 and NALM-6 cell lines treated for 24 hours with increasing concentrations of PF-00477736 (B). Figure S4. Western Blot analysis in all leukemia cell lines after exposure to PF-00477736 (+) at the concentration closest the IC50 or DMSO 0.1 %(-) (A). Western Blot analysis of BV-173 and NALM-6 cell lines treated with or without PF-00477736 (IC50) for 3, 6 and 9 hours (B). Figure S5. Cell cycle_ESR1 regulation of G1/S transition: the top scored map (map with the lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S6. Apoptosis and survival_Granzyme A signaling: the second scored map (map with the second lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S7. DNA damage_ATM/ATR regulation of G1/S checkpoint: the third scored map (map with the third lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S8. Protein expression level of c-Jun in B-/T-ALL cell lines (A). Western Blot analysis of normal MNCs and ALL MNCs after exposure to PF-0047773(B). mRNA expression of CDK4, Chk2, GADD45a and PLK3 in B-/T-ALL cell lines treated with or without PF-00477736(IC50 value) for 24 hours. The results are expressed as 2exp(-ΔΔCt)(C). (PPTX 2905 kb)

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.

Table S1. Patient’s characteristic of gene expression cohort. Table S2. Patient’s characteristic ex vivo AZD-1775 treatment in single agent or in combination. Table S3. Quantitative analyses of G2/M checkpoint-related genes. Differential gene expression of 24 genes involved in the regulation of the G2/M checkpoint of primary leukemic cells in comparison to normal mononuclear cells (MNCs). In the table, the primary leukemic samples have been divided into three groups based on the ex vivo sensitivity to AZD-1775. Very good IC50 10 uM. (PDF 253 kb)

Table S1. Patient’s characteristic of gene expression cohort. Table S2. Patient’s characteristic ex vivo AZD-1775 treatment in single agent or in combination. Table S3. Quantitative analyses of G2/M checkpoint-related genes. Differential gene expression of 24 genes involved in the regulation of the G2/M checkpoint of primary leukemic cells in comparison to normal mononuclear cells (MNCs). In the table, the primary leukemic samples have been divided into three groups based on the ex vivo sensitivity to AZD-1775. Very good IC50 10 uM. (PDF 253 kb)

Figure S1. Efficacy of AZD-1775 used as single agent. A) The graph shows the IC50 values of B/T-ALL cell lines treated with AZD-1775 for 24, 48, and 72 h. B) Cell viability analysis on CCRF-CEM cell lines showing the effect of high doses of AZD-1775. The percentage of viable cells is depicted relative to untreated controls. C) Immunoblot analysis on BV-173 treated with AZD-1775 (IC50) for 12 h. D) Cell cycle analysis in BV-173 and CCRF-CEM cell lines treated with AZD-1775 (IC50) for 12 h. E) Immunofluorescence analysis of BV-173 cells treated with AZD-1775 (IC50) for 12 h and, then, stained with DAPI and phospho-MPM2. In the picture, a cell dying in mitosis is reported with apoptotic bodies strongly positive for phospho-MPM2 antibody. Representative images are shown at × 100 magnification. F) Viability of mononuclear cells isolated from the peripheral blood of 5 healthy donors incubated with increasing concentration of AZD-1775 (2.5, 5, and 10 uM) for 24 h. G) MYT1 transcript levels in samples isolated from adult BCR-ABL1-positive ALL at diagnosis (n = 17), adult BCR-ABL1-negative ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood of healthy donors. One-way ANOVA test was performed to assess statistical significance. Results are expressed as Log10 2 exp.[−(ΔΔCt). Figure S2. AZD-1775 in combination with chemotherapy agents and tyrosine kinase inhibitors. A) Growth curve of BV-173 and REH cell lines treated for 4 days with AZD-1775 (185 nM) and doxorubicin (25 nM). B) Viability analyses in ALL cell lines incubated for 24 h with Bos or Bos-I (6 to 5000 nM). The percentage of viable cells is depicted relative to untreated controls. C) Cell viability analysis of BV-173 cell line treated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with ponatinib (25, 50, 100 nM) or imatinib (250, 500, and 1000 nM) for 24 h. The percentage of viable cells is depicted relative to untreated controls, 0.1%. Figure S3. Wee1 mRNA expression across different cancer types from the Cancer Cell Line Encyclopedia (CCLE) database. A) Box plots showing the level of expression of Wee1 mRNA in different tumor samples, extracted from CCLE [63]. The red arrows point to B/T-ALL samples. Boxes define the 25th and the 75th percentiles, horizontal line within the boxes indicates the median, and whiskers define the 10th and the 90th percentiles. (PDF 1918 kb)

Figure S1. Efficacy of AZD-1775 used as single agent. A) The graph shows the IC50 values of B/T-ALL cell lines treated with AZD-1775 for 24, 48, and 72 h. B) Cell viability analysis on CCRF-CEM cell lines showing the effect of high doses of AZD-1775. The percentage of viable cells is depicted relative to untreated controls. C) Immunoblot analysis on BV-173 treated with AZD-1775 (IC50) for 12 h. D) Cell cycle analysis in BV-173 and CCRF-CEM cell lines treated with AZD-1775 (IC50) for 12 h. E) Immunofluorescence analysis of BV-173 cells treated with AZD-1775 (IC50) for 12 h and, then, stained with DAPI and phospho-MPM2. In the picture, a cell dying in mitosis is reported with apoptotic bodies strongly positive for phospho-MPM2 antibody. Representative images are shown at × 100 magnification. F) Viability of mononuclear cells isolated from the peripheral blood of 5 healthy donors incubated with increasing concentration of AZD-1775 (2.5, 5, and 10 uM) for 24 h. G) MYT1 transcript levels in samples isolated from adult BCR-ABL1-positive ALL at diagnosis (n = 17), adult BCR-ABL1-negative ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood of healthy donors. One-way ANOVA test was performed to assess statistical significance. Results are expressed as Log10 2 exp.[−(ΔΔCt). Figure S2. AZD-1775 in combination with chemotherapy agents and tyrosine kinase inhibitors. A) Growth curve of BV-173 and REH cell lines treated for 4 days with AZD-1775 (185 nM) and doxorubicin (25 nM). B) Viability analyses in ALL cell lines incubated for 24 h with Bos or Bos-I (6 to 5000 nM). The percentage of viable cells is depicted relative to untreated controls. C) Cell viability analysis of BV-173 cell line treated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with ponatinib (25, 50, 100 nM) or imatinib (250, 500, and 1000 nM) for 24 h. The percentage of viable cells is depicted relative to untreated controls, 0.1%. Figure S3. Wee1 mRNA expression across different cancer types from the Cancer Cell Line Encyclopedia (CCLE) database. A) Box plots showing the level of expression of Wee1 mRNA in different tumor samples, extracted from CCLE [63]. The red arrows point to B/T-ALL samples. Boxes define the 25th and the 75th percentiles, horizontal line within the boxes indicates the median, and whiskers define the 10th and the 90th percentiles. (PDF 1918 kb)

Mantle cell lymphoma (MCL) usually harbors the t(11;14)(q13;q32) with overexpression of CCND1 mRNA and transcription of the cyclin D1 nuclear protein. Regardless of CCND1 status, most MCLs also express the SOX11 nuclear protein, which is thus helpful in the diagnosis of the rare CCND1-negative MCLs. Recently, SOX11 has been reported to be often negative in MCLs clinically resembling marginal zone lymphoma and recently defined as “leukemic non-nodal” MCL in the incoming revision of the WHO classification of lymphoid tumors, for which the bone marrow biopsy is commonly the first diagnostic approach. Due to the less aggressive clinical behavior of the latter MCLs, the reliable determination of the SOX11 antigen in decalcified tissue is mandatory. To this end, since little data are available in the literature, four commercially available anti-SOX11 antibodies (two polyclonal and two monoclonal) were tested on 21 positive staging bone marrow (BM) biopsies from cyclin D1/SOX11–positive MCL patients (17 fixed in B5, 4 in 10% buffered formalin) and on 9 positive BM biopsies from leukemic non-nodal MCL patients. The results were compared for specificity, sensitivity, staining strength and degree of an additional staining on myeloid precursors, also evaluating possible impact of the different fixatives used. Non-mantle cell lymphomas were also tested to address specificity. All reagents showed high sensitivity but the monoclonal code CMC38221001 provided the highest specificity and the lowest degree of non-lymphoid staining on myeloid cells. Formalin fixation generally improved the performance of most antibodies when compared to B5 fixation.

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.

// Enrico Derenzini 1 , Claudio Agostinelli 2 , Enrica Imbrogno 1 , Ilaria Iacobucci 1 , Beatrice Casadei 1 , Elisa Brighenti 1 , Simona Righi 2 , Fabio Fuligni 2 , Andrea Ghelli Luserna Di Rora 1 , Anna Ferrari 1 , Giovanni Martinelli 1 , Stefano Pileri 2 , Pier Luigi Zinzani 1 1 Institute of Hematology and Medical Oncology L.A. Seragnoli, Department of Experimental, Diagnostic and Specialty Medicine - DIMES, University of Bologna, Italy 2 Haematopathology Unit, Department of Experimental, Diagnostic and Specialty Medicine - DIMES, University of Bologna, Italy Correspondence to: Enrico Derenzini, e-mail: enrico.derenzini3@unibo.it Keywords: Diffuse Large B-cell Lymphoma, genomic instability, CHK1, CHK2, gamma-H2AX, MYC Received: August 23, 2014 Accepted: November 08, 2014 Published: January 07, 2015 ABSTRACT The recent finding that MYC -driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon-inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.