Patient-specific Gene Expression Research Articles

Single Cell Multi-Omic Profiling of Acute Lymphoblastic Leukemia Identifies Altered Cellular Pathways and Highlights Heterogeneity in the Maturation Block of DUX4-Rearranged ALL

B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. The established subtypes of BCP-ALL, such as BCR::ABL1-positive, ETV6::RUNX1-positive, and high hyperdiplod (HeH) ALL, are important for diagnosis, prognostication, and treatment stratification. However, we and others have described novel subtypes, of which DUX4-rearranged (DUX4-r) ALL is among the most common. The subtype-specific rearrangements of DUX4 result in aberrant expression of a truncated version of this gene, which normally encodes an embryonic transcription factor. ALL is typically associated with a good prognosis, and DUX4-r ALL represents a favorable subtype. However, the cytotoxic treatment is associated with significant short- and long-term side effects. By an increased understanding of the cellular and molecular characteristics of ALL, the development of more specific treatment alternatives with high efficacy and less toxicity should be facilitated. We have used a multimodal single cell approach to delineate the transcriptional, epigenetic and phenotypic characteristics of a cohort of childhood BCP-ALLs (n=23) with a focus on the novel DUX4-r ALL subtype (n=11). The cohort also included three established subtypes, with four samples each of BCR::ABL1, ETV6::RUNX1, and HeH. The leukemic blast cell populations of the DUX4-r cases (blast count at diagnosis: 70 - 96 %) displayed distinct gene expression profiles. To deduce where the blast cells were located along a normal differentiation axis, we projected the blast cells onto a reference force directed knn-graph of normal bone marrow (NBM) cells from five healthy donors (Fig. 1). The projection of cells allowed inferring the most likely stage of maturation for each cell, as well as visualizing the altered gene expression profile compared to their healthy counterparts using a "dissimilarity score". Several DUX4-r ALL (4/11) samples exhibited a lack of B-cell progenitors after a distinct point in the graph, suggesting a distinct maturation block. For the remaining samples, the blast cells were dispersed throughout the B-cell maturation axis, indicating a partial or a more heterogenous arrest among the leukemic blasts. We also classified the cell type of each cell in the ALL samples according to a NBM reference. With this method, most of the blast cells were characterized as early progenitor B-cells. However, other cell classifications also occurred. In fact, in five of eleven DUX4-r ALLs, more than 35% of the blast population were classified as a cell type distinct from B-cell progenitors. Interestingly, the classification of blast cells as granulocyte-macrophage progenitors (GMP) was particularly common among DUX4-r ALLs (2-30% of blast cells) compared to the other subtypes (0-12%). However, such cells displayed an altered gene expression profile compared to GMP cells in NBM, as manifested by a high dissimilarity score. Nevertheless, this could suggest a higher plasticity or a more myeloid-biased transcriptional state among the blast cells within the DUX4-r subtype compared to other subtypes. To further delineate altered pathways in DUX4-r blast cells, differential gene expression analyses were performed, both between DUX4-r blast cells and blast cells of other subtypes, as well as between DUX4-r blast cells and NBM progenitor B-cells. The DUX4-r blast cells were characterized by high expression of distinct outlier genes, including ANGPT2, MCAM, EGR1, PDGFRA, NR4A1, AFT3 and PTPRM. Notably, GSEA highlighted upregulation of gene sets involved in angiogenesis- and vasculature development. In conclusion, by single cell multi-omic profiling we demonstrate that BCP-ALL blast cells are associated with distinct patient-specific gene expression patterns that are strongly influenced by subtype. DUX4-r ALL blast cells are associated with a heterogenous maturation block, transcriptional features associated with angiogenesis- and vasculature development, and possibly also higher plasticity with a myeloid-biased transcriptional state. The results should, hopefully, form a basis towards improved future diagnostics and treatment stratification of BCP-ALL, and aid the discovery of targeted treatment alternatives. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of Single Cell Transcriptomics in Paired Pediatric T-ALL Samples Collected at Diagnosis and Following End of Induction Therapy Reveals an MRD-Associated Stem Cell Signature

IntroductionDespite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups.MethodsUsing the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021).ResultsUsing our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B).Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. [Display omitted] DisclosuresDeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

A Personalized Therapeutics Approach Using an In Silico Drosophila Patient Model Reveals Optimal Chemo- and Targeted Therapy Combinations for Colorectal Cancer.

In silico models of biomolecular regulation in cancer, annotated with patient-specific gene expression data, can aid in the development of novel personalized cancer therapeutic strategies. Drosophila melanogaster is a well-established animal model that is increasingly being employed to evaluate such preclinical personalized cancer therapies. Here, we report five Boolean network models of biomolecular regulation in cells lining the Drosophila midgut epithelium and annotate them with colorectal cancer patient-specific mutation data to develop an in silico Drosophila Patient Model (DPM). We employed cell-type-specific RNA-seq gene expression data from the FlyGut-seq database to annotate and then validate these networks. Next, we developed three literature-based colorectal cancer case studies to evaluate cell fate outcomes from the model. Results obtained from analyses of the proposed DPM help: (i) elucidate cell fate evolution in colorectal tumorigenesis, (ii) validate cytotoxicity of nine FDA-approved CRC drugs, and (iii) devise optimal personalized treatment combinations. The personalized network models helped identify synergistic combinations of paclitaxel-regorafenib, paclitaxel-bortezomib, docetaxel-bortezomib, and paclitaxel-imatinib for treating different colorectal cancer patients. Follow-on therapeutic screening of six colorectal cancer patients from cBioPortal using this drug combination demonstrated a 100% increase in apoptosis and a 100% decrease in proliferation. In conclusion, this work outlines a novel roadmap for decoding colorectal tumorigenesis along with the development of personalized combinatorial therapeutics for preclinical translational studies.

Differential regulation of a placental SAM68 and sFLT1 gene pathway and the relevance to maternal vitamin D sufficiency.

The goal of this study was to determine if an axis of placental gene expression associated with early onset and severe preeclampsia (EOSPE) was operative in term pregnancy and correlated with vitamin D sufficiency. qPCR analysis of NKX2-5, SAM68, sFLT1 and membrane bound VEGFR1/FLT1 mRNA expression was conducted in placentas from 43 subjects enrolled in a vitamin D3 pregnancy supplementation trial. Pair-wise rank order correlations between patient-specific gene expression levels were calculated, and their relationship to maternal 25(OH)D status was assessed by a two-sample Wilcoxon test. Additionally, we probed the mechanistic link between SAM68 and sFLT1 using siRNA depletion in a human trophoblast cell line model. Positive and highly significant correlations were found between SAM68 vs. sFLT1 and SAM68 vs. FLT1 expression levels, as were significant and differential correlations between the expression of these genes and perinatal 25(OH)D status. The variability when stratified by race/ethnicity was qualitatively distinct from those previously observed in EOSPE. Mechanistic studies confirmed a functional role for SAM68 protein in the regulation of sFLT1 expression. NKX2-5 expression was not significantly correlated with sFLT1 or SAM68 expression in these samples, suggesting that its expression may be significant at earlier stages of pregnancy or be restricted to pathological settings. These data further support our overarching hypothesis that SAM68 expression is a key determinant of VEGFR1 isoform expression in the placenta, and provide additional insights into how this gene pathway may be differentially deployed or modified in normal and pathological pregnancies.

Single-Cell RNA Sequencing Identifies a Pseudo-Immune Differentiation Axis As the Main Source of Functional Heterogeneity in Follicular Lymphoma B-Cells

Introduction Follicular Lymphoma (FL), the second most frequent lymphoma in adults, often presents as a disseminated disease at diagnosis. Despite a generally slow progression and a median overall survival of more than 15 years with current chemo-immunotherapies, FL patients often suffer from multiple relapses. Yet, the biological mechanisms promoting FL dissemination, progression and relapse are still poorly understood. FL, like most B-cell lymphomas, originates from germinal centers (GC) where B-cells physiologically undergo clonal expansion, antibody affinity maturation, and differentiation into antibody-producing plasma cells (PC) or recirculating memory (Mem) B-cells. Recently, we provided evidence that FL B-cells are not blocked in a GC B-cell state but might adopt new dynamic modes of functional diversity (Milpied et al., Nature Immunology 2018), yet the main sources of intratumoral heterogeneity within FL remained to be identified. Methods Frozen live cell suspensions were obtained from the CeVi collection of the Institute Carnot/Calym (ANR, France). We initially applied a plate-based 5'-end single-cell RNAseq (scRNAseq) method for deep integrative single-cell analyses of transcriptome, B-cell receptor (BCR) sequence, and surface phenotype on FACS-sorted FL B-cells (4 patients, lymph node biopsies) and their non-malignant counterparts (6 adult healthy donors, spleen and tonsil samples). We confirmed our findings on additional FL samples with high-throughput droplet-based 3'-end scRNAseq (9 patients, lymph node biopsies), and 5'-end scRNAseq paired with BCRseq (5 patients, lymph node biopsies). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE, UMAP), clustering, pseudo-time analysis, BCR sequence analysis and integrative data analysis. We further validated our transcriptomic data with FACS-based surface and intracellular protein analysis (8 patients, lymph node biopsies). Results Consistent with our previous findings, FL B-cells were transcriptionally diverse, with most cells exhibiting a patient-specific gene expression profile distinct from PC, GC and Mem cells. Challenging the mainstream view of a differentiation blockade in FL, we identified rare FL B-cells carrying a PC-like profile (including low expression of MS4A1/CD20, high expression of XBP1, MZB1, PRDM1). PC-like FL B-cells expressed high levels of the tumor clonal BCR heavy and light chain mRNA, and BCR sequence phylogenetic analysis revealed that those cells did not branch out from a specific tumor subclone. Most importantly, we found that the molecular profiles of the vast majority of FL B-cells spanned a continuum of transitional states between proliferating GC-like and quiescent Mem-like gene expression states. Principal component analysis and pseudo-time reconstruction revealed that pseudo-immune differentiation axis was consistently the main source of intra-sample transcriptional heterogeneity. On top of cell cycle related genes, GC-like FL B-cells notably expressed AICDA, BCL6, RGS13, NANS, CD81, and CD38 genes. By contrast, Mem-like FL B-cells expressed CD44, GPR183, CD69, CXCR4, CCR7, SELL, KLF2, suggesting that those cells may not be confined to the FL follicles. Flow cytometry analysis of dissociated FL tumors confirmed that only the CD38hiCD81hi subset of FL B-cells (GC-like cells), expressed Ki67 and high levels of Bcl6, whereas only CD38negCD81neg FL B-cells (Mem-like cells) consistently contained CD44+ and GPR183+ cells. Conclusions Our study suggests that FL B-cells hijack the physiological GC differentiation process to dynamically alternate between GC-like and Mem-like states that might be responsible for FL progression and dissemination, respectively. We anticipate that such FL-specific clonal dynamics may be orchestrated by extrinsic signals delivered by tumor-infiltrating T cells. Disclosures Milpied: Innate Pharma: Research Funding; Institut Roche: Research Funding.

Gene expression profile of peripheral blood mononuclear cells may contribute to the identification and immunological classification of breast cancer patients.

It has been reported that the gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits a unique gene expression signature in several types of cancer. In this study, we aimed to explore the breast cancer patient-specific gene expression profile of PBMCs and discuss immunological insight on host antitumor immune responses. We comprehensively analyzed the gene expression of PBMCs by RNA sequencing in the breast cancer patients as compared to that of healthy volunteers (HVs). Pathway enrichment analysis was performed on MetaCoretm to search the molecular pathways associated with the gene expression profile of PBMCs in cancer patients compared with HVs. We found a significant unique gene expression signature, such as the Toll-like receptor (TLR) 3- and TLR4-induced Toll/interleukin-1 receptor domain-containing adapter molecule 1 (TICAM1)-specific signaling pathway in the breast cancer patients as compared to that of healthy volunteers. Distinctive immunological gene expression profiles also showed the possibility of classifying breast cancer patients into subgroups such as T-cell inhibitory and monocyte-activating groups independent of known phenotypes of breast cancer. These preliminary findings suggest that evaluation of gene expression patterns of PBMCs might be both a less invasive diagnostic procedure and a useful way to reveal immunological insight of breast cancer, including biomarkers for cancer immunotherapy, such as immune checkpoint inhibitor therapy.

Abstract 416: Identification of therapeutic combinations in glioblastoma using personalized gene expression networks

Abstract The goal of our study was to identify patient-specific gene expression networks from Glioblastoma Patient-Derived Xenografts (PDXs) and determine novel therapeutic compound combinations using those networks. Glioblastoma is the most common malignant primary adult brain tumor with a standard of care consisting of maximal surgical resection followed by radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. However, despite medical advances in the field, recurrence is almost universal, suggesting the need for more personalized and targeted therapeutic approaches. For this, we obtained, transcriptional data from Glioblastoma PDXs and used them to identify their respective differentially expressed genes. Patient-specific gene expression networks were then created and their biological relevance was supplemented by integrating them with TCGA Glioblastoma transcriptional data. In order to identify compound combinations specific for those networks, we used the extensive chemical perturbation signatures from the Library of Network-based Cellular Signatures (LINCS). From the large number of L1000 transcriptional data we extracted gene expression signatures that were indicative of specific LINCS compounds. We then compared those signatures to the patient-specific networks in order to prioritize compound combinations that were inducing discordant transcriptional changes in distinct sub-networks of the PDXs transcriptome. The most orthogonal compound combinations were then chosen and used in in-vitro cell viability assays of Glioblastoma PDXs in order to evaluate their effectiveness. The above process can be used to prioritize compound combinations with potential therapeutic effect in a patient-specific manner. Citation Format: Vasileios Stathias, Michele Forlin, Bryce Allen, Stephan Schürer, Nagi G. Ayad. Identification of therapeutic combinations in glioblastoma using personalized gene expression networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2017-416

Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In Vitro

Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patientvariation than intra-patient variation, verifyingthat reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used invitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.

Abstract A39: Patient-derived xenografts to test emerging therapies for triple negative breast cancer

Abstract Introduction: Patients with triple negative breast cancer (TNBC) that do not respond to cytotoxic neoadjuvant therapy have a poor prognosis, thus there is a pressing need to develop pre-clinical models of chemoresistant TNBC and identify optimal therapeutic targets. Patient-derived xenografts (PDX) are a simple, expandable approach to model TNBC in vivo. We have generated a collection of triple negative PDXs that have undergone molecular characterization at the DNA, RNA and protein levels and have initiated characterization of their therapeutic sensitivity to standard chemotherapeutics regimens as well as targeted therapies. Methods: Tumors from patients and matching PDXs have been characterized by cancer-specific targeted exome sequencing, RNA sequencing, and Reverse Phase Protein Arrays. We have established single agent sensitivities for a set of PDXs to standard TNBC therapies (paclitaxel, carboplatin, doxorubicin, eribulin, gemcitabine) and the following targeted therapies: talazoparib (PARP), MLN0128 (mTOR), buparlisib (PI3K), and trametinib (MEK1/2). Results: We have generated 25 hormone receptor-low/HER2- PDXs from 24 patients (1 primary-local recurrence pair) with an overall engraftment rate of 49% for this patient population. Exome sequencing found that the PDXs mostly maintain the somatic mutation profile of the originating tumor and exhibit alterations common to TNBC: 16 (73%) with TP53 mutations, 2 (9%) with PI3KCA mutations and 4 (18%) with PTEN deletions. RNA sequencing demonstrated that the PDXs maintain gene expression profiles similar to the originating tumor and that our panel is comprised of PDXs representative of five TNBC molecular subtypes. Treatment with standard therapies established differential sensitivity to paclitaxel and carboplatin between PDXs. The two models with PI3KCA alterations were responsive to mTOR/PI3K inhibition and unresponsive to trametinib and talazoparib, supporting heightened reliance on mTOR/PI3K signaling in PI3KCA mutant TNBC. Talazoparib was the only targeted agent tested that caused regression as a single agent in any model. All three models that regressed with talazoparib treatment had genetic alterations linked to DNA damage pathways (BRCA1 germline alteration, ATM deletion; PTEN deletion); these models also had low BRCA1 mRNA expression and were the only PDXs to regress when treated with carboplatin. Conclusions: PDXs of all TNBC subtypes can be generated from TNBC patients that are resistant to neoadjuvant chemotherapy. These PDXs maintain molecular alterations of TNBC, retain patient-specific gene expression, and display differential response to therapies enabling identification of associations between responses and molecular traits. Citation Format: Kurt W. Evans, Erkan Yuca, Stephen M. Scott, Natalia Arango Paez, Xiaofeng Zheng, Ken Chen, Laura Gonzales, Coya Tapia, Emily Tarco, Dalliah M. Black, Gordon B. Mills, Funda Meric-Bernstam. Patient-derived xenografts to test emerging therapies for triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A39.

Abstract 289: Transcriptomic Analysis of Inter- and Intra-patient Variation in Human iPSCs: Platform for Precision Medicine to Predict Drug Toxicity

Rapid improvements in human induced pluripotent stem cell (hiPSC) differentiation methodologies have allowed previously unattainable access to high-purity, patient-specific cardiomyocytes (CMs) for use in disease modeling, cardiac regeneration, and drug testing. In the present study, we investigate the ability of hiPSC-derived cardiomyocytes (hiPSC-CMs) to reflect the donor’s genetic identity and serve as preclinical functional readout platforms for precision medicine. We used footprint-free Sendai virus to create two separate hiPSC clones from the fibroblasts of five different individuals lacking known mutations associated with cardiovascular disease. Whole genome expression profiling of hiPSC-CMs showed that inter-patient variation was greater than intra-patient variation, thereby verifying that reprogramming and cardiac differentiation technologies can preserve patient-specific gene expression signatures. Gene ontologies (GOs) accounting for inter-patient variation were mostly metabolic or epigenetic. Toxicology analysis based on gene expression profiles predicted patient-specific susceptibility of hiPSC-CMs to cardiotoxicity, and functional assays using drugs targeting key regulators in pathways predicted to produce cardiotoxicity showed inter-patient differential responses in hiPSC-CMs. Our data suggest that hiPSC-CMs can be used in vitro to predict and help prevent patient-specific drug-induced cardiotoxicity, potentially enabling personalized patient consultation in the future.

Panobinostat As Salvage Treatment for Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma Not Eligible to High Dose Therapy: A Phase II Study of the Fondazione Italiana Linfomi (FIL)

Abstract Backgrounds: Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Histone deacetylases (DACs) regulate chromatin structure and function and are involved in crucial mechanisms of lymphoma cell growth. Panobinostat showed encouraging therapeutic activity in Hodgkin lymphoma, cutaneous T-cell lymphoma and other non-Hodgkin lymphomas, in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies. Moreover, recent studies showed a potent activity of Panobinostat in DLBCL. Purpose: On this basis we performed a prospective, multicenter, phase II single arm study, to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL adult patients and to evaluate a possible relationships between response and any biological features. Patients and Methods: Adult patients with R/R DLBCL who already performed high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) or were not eligible for ASCT were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation; patients achieving complete response (CR), partial response (PR) or stable disease (SD), underwent maintenance for a maximum of 36 courses. In each 28-days course, Panobinostat was administered orally at the dosage of 40 mg/day three-times every week; dose adjustments for patients unable to tolerate the protocol-specified schedule were provided. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR) according to the Cheson 1999 criteria; secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient's specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. Results Thirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. Clinical characteristics were: median age 73 (range 65-75), stage IV in 18 (55%), B-symptoms in 9 (28%), increased LDH in 24 (69%), high-intermediate or high International Prognostic Index (IPI) in 18 (51%). Patients received a median of 2 prior lines of therapy (range 1-4). At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. In univariate analysis, favourable IPI score and cutaneous involvement at enrollment showed a trend toward a higher ORR (p=0.007 and 0.061, respectively); pharmacogenetics, immunohistochemical and gene expression profile studies are still ongoing. No toxic deaths were reportewd; 18 patients died, 17 due to lymphoma progression and one for allogeneic transplant related complications, performed after PD. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively), while grade 3-4 extra-hematological toxicity included diarrhoea in 4 (12%), infectious complications in 1 (3%) and supraventricular arrhythmia in 2 patients (6%). Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR. Feasibility was impaired by relevant hematological toxicity, mainly frequent and dose limiting grade 3-4 thrombocytopenia. Data that will be obtained from biological exploratory studies could hopefully be useful to better address the use of Panobinostat in peculiar subsets of patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Off Label Use: Panobinostat in DLBCL.

CD34+ gene expression profiling of individual children with very severe aplastic anemia indicates a pathogenic role of integrin receptors and the proapoptotic death ligand TRAIL

BACKGROUND Very severe aplastic anemia is characterized by a hypoplastic bone marrow due to destruction of CD34(+) stem cells by autoreactive T cells. Investigation of the pathomechanism by patient-specific gene expression analysis of the attacked stem cells has previously been impractical because of the scarcity of these cells at diagnosis. Employing unbiased RNA amplification, patient-specific gene expression profiling was carried out for CD34(+) cells from patients newly diagnosed with very severe aplastic anemia (n=13), refractory anemia (n=8) and healthy controls (n=10). These data were compared to profiles of myelodysplastic disease (n=55), including refractory anemia (n=18). To identify possible targets of autoimmune attack, presence of autoreactive antibodies was tested in pre-therapeutic sera of patients with very severe aplastic anemia (n=19). CD34(+) gene expression profiling distinguished between healthy controls, children with aplastic or refractory anemia and clonal disease. Interferon stimulated genes such as the apoptosis inducing death ligand TRAIL were strongly up-regulated in CD34(+) cells of patients with aplastic anemia, in particular in patients responding to immunosuppressive treatment. In contrast, mRNA expression of integrin GPVI and the integrin complexes GPIa/IIa, GPIIb/IIIa, GPIB/GPIX/GPV was significantly down-regulated and corresponding antibodies were detected in 7 of 11 profiled patients and in 11 of 19 aplastic anemia patients. CONCLUSIONS As a potential diagnostic tool, patient-specific gene expression profiling of CD34(+) stem cells made it possible to make the difficult differential diagnosis of most patients with aplastic and refractory anemia. Profiling indicated a prognostic correlation of TRAIL expression and patient benefit from immunosuppressive therapy. Downregulation of integrin expression and concurrent presence of autoreactive anti-integrin-antibodies suggested a previously unrecognized pathological role of integrins in aplastic anemia.

Disease profiling arrays: reverse format cDNA arrays complimentary to microarrays.

The identification of differentially expressed genes enables us to understand the molecular mechanisms associated with disease, conditions of stress, drug treatments and developmental processes. Microarrays provide a powerful tool for studying these complex phenomena. Verification of differentially expressed genes and correlation with biological function, which is usually done by northern blot analysis, RNase protection assay or RT-PCR, is the bottleneck in all these protocols. We developed a new type of cDNA array for high-throughput expression profiling of multiple tissues and blood samples (i) for confirmation analysis of statistically significant number of clinical samples (ii) from limited amount of starting material, and (iii) with detailed clinical data from each individual. In contrast to traditional cDNA arrays, these arrays are spotted with a complex cDNA representing the entire mRNA message expressed in a given tissue or blood sample. cDNAs for these arrays were generated using SMART technology and accurately represent the original mRNA population, producing specific and quantitative signals during hybridization. cDNAs on Disease Profiling Arrays were derived from total RNAs of diseased and normal tissues or different blood fractions of patients. These cDNAs were spotted onto nylon membranes along with positive and negative controls. The arrays were then hybridized with gene-specific probes. Hybridization results revealed disease-related as well as patient-specific gene expression patterns between different disease types for these genes. These studies demonstrate that Disease Profiling Arrays are suitable for high-throughput studies comparing the relative abundance of a target gene, for simultaneously detecting differentially expressed genes in a wide variety of tissues and blood samples, and can be used down-stream from cDNA microarrays for confirmation analysis.

Use of SMART-generated cDNA for gene expression studies in multiple human tumors.

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.

Impaired acetaldehyde metabolism in patients with non-alcoholic liver disorders.

In order to determine the specificity of abnormalities of alcohol metabolism in patients with alcoholic liver disease, blood acetaldehyde concentrations after oral ethanol challenge and the activities of alcohol metabolising enzymes in liver biopsy samples have been determined in patients with alcoholic liver disease and a wide variety of non-alcoholic liver disorders. Significant decreases in hepatic cytosolic aldehyde dehydrogenase activity were associated with significant increases in acetaldehyde concentrations after ethanol in both patient groups compared with control subjects. There was a significant correlation between hepatic cytosolic aldehyde dehydrogenase and mean blood acetaldehyde concentration 30-180 min after ethanol ingestion (y = 17.4-0.45x; r = -0.56; p less than 0.01) confirming the importance of this enzyme in controlling blood acetaldehyde concentrations. These findings suggest that disturbances in alcohol metabolism in patients with alcoholic liver disease are the consequence of liver damage rather than a specific abnormality predisposing to alcohol induced liver injury.