A multilevel data integration resource for breast cancer study

BACKGROUND: Breast cancer is one of the most common cancer types. Due to the complexity of this disease, it is important to face its study with an integrated and multilevel approach, from genes, transcripts and proteins to molecular networks, cell populations and tissues. According to the systems biology perspective, the biological functions arise from complex networks: in this context, concepts like molecular pathways, protein-protein interactions (PPIs), mathematical models and ontologies play an important role for dissecting such complexity. RESULTS: In this work we present the Genes-to-Systems Breast Cancer (G2SBC) Database, a resource which integrates data about genes, transcripts and proteins reported in literature as altered in breast cancer cells. Beside the data integration, we provide an ontology based query system and analysis tools related to intracellular pathways, PPIs, protein structure and systems modelling, in order to facilitate the study of breast cancer using a multilevel perspective. The resource is available at the URL http://www.itb.cnr.it/breastcancer. CONCLUSIONS: The G2SBC Database represents a systems biology oriented data integration approach devoted to breast cancer. By means of the analysis capabilities provided by the web interface, it is possible to overcome the limits of reductionist resources, enabling predictions that can lead to new experiments

A challenging case of fever of unknown origin (FUO)

Introduction. Fever of unknown origin (FUO) is challenging for physicians as there are more than 200 differential diagnosis of FUO. The diagnosis often requires numerous non-invasive and invasive procedures and sometimes the etiology remains unknown. Clinical case. Our patient is a 24-year-old Caucasian female under contraceptive vaginal ring treatment who presented the first time to the emergency department with fever (38,5°C) and stomach ache. Haematological parameters showed thrombocytosis (728000/mmc) and anemia (10,2 g/dl). Ultrasonography of the abdomen revealed a 4 cm hepatic angioma. She was diagnosed a gastropathy and treated with PPI. Then she was studied deeper because of elevated inflammation markers, persistent thrombocytosis and low-grade fewer combined with fatigue and stomach ache. Despite multiple blood and urine cultures, serology, autoimmune serology, bone marrow biopsy, echocardiography, PET/CT, total body CT, brain MRI scan, transvaginal sonography and gynecological examination no clinical focus could be identified. Esophagogastroduodenoscopy was negative but the histological examination was suggestive of mild celiac disease. Thus, this diagnosis was unlikely because serologic tests and haplotypes DQ2 and DQ8 were negative. Abdomen RMI was suggestive of a 4 cm hepatic adenoma. After surgical resection of the adenoma platelet count normalized and fever disappeared. Conclusions. Hepatic adenoma is an uncommon liver tumor associated with use of oral contraceptive and can be a rare cause of FUO. MRI scan can be a useful tool to detect this tumor

530. Development of New Lentiviral Vectors With a Reduced Splicing Interference Potential and a Safer In Vivo Genotoxic Profile

The excellent therapeutic potential of self-inactivating (SIN) lentiviral vectors (LV) has been demonstrated in pre-clinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could endanger their clinical applications. Systemic vector injection into newborn tumor-prone Cdkn2a-/- and Cdkn2a+/-mice, conducted in our previous work, demonstrated that SINLVs harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset with respect to control mice. Integration sites analyses of vector-induced tumor showed that oncogene activations or tumor suppressor inactivation by LV integrations occur by combining mechanisms of transcript truncation, induction of aberrant splicing and/or enhancer-mediated overexpression of cellular transcription units. Although oncogene activation may be reduced by the use of self-inactivating design, moderate cellular promoters and insulator sequences how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear.From this and a previous study, we identified the LV sequences most frequently involved in chimeric transcript formation. In our rationale, these LV sequences could be tagged by sequences complementary to microRNAs (mirT sequence) active in hematopoietic cells in order to allow selective degradation, through the miRNA pathway, of vector-mediated aberrantly spliced transcripts. Hence, we specifically designed SIN LVs harboring mirT sequences recognized by mir223 and mir142-3p (that are expressed in hematopoietic lineages) within the SIN LTR (mirsT-LTR LV) or in the vector backbone and outside the gene expression cassette (mirT LV). We then assessed the genotoxicity of the SIN LVs harboring mirT sequences by taking advantage of our in vivo models. Interestingly, injection of mirsT-LTR LV (N=73) and mirT LV (N=73) in Cdkn2a-/- mice did not caused any significant acceleration in hematopoietic tumor onset compared to control un-injected mice (N=40). Similar results have been obtained after injection in Cdkn2a+/- mice (N=28 for mirsT-LTR LV, N=26 for mirT LV and N=34 un-injected mice). We are currently performing integration site analyses in Cdkn2a-/- and Cdkn2a+/- treated mice to dissect if and how the integrated mirsT-LTR LV and mirT LV proviral genome interacts with the surrounding cellular genome.Overall, these studies show that this new advanced design lentiviral vectors completely abrogated residual vector genotoxicity in highly sensitive mouse models and could represent the vector design of choice in future gene therapy applications

535. Increasing Accuracy and Precision of Vector Integration Site Identification of Sequencing Reads With a New Bioinformatics Framework

In hematopoietic stem cell (HSC) gene therapy (GT) applications patients are transplanted with autologuos HSCs that have been ex-vivo genetically modified with integration competent vectors to express a therapeutic transgene. Specific PCR techniques coupled to next generation sequencing and bioinformatics analysis allow the high throughput retrieval, sequencing and mapping of proviral/genomic DNA junctions present in the blood and bone marrow derived cell populations sampled at different time points after therapy. The increase in sequences available for IS mapping is accompanied by an increase in false positives derived by sequencing errors or sequencing read parsing and mapping on the reference genome. In particular, by analyzing IS datasets form vector marked human and mouse tumor cells, clones with defined integration sites and GT patients, we observed that when multiple sequences arising from the same IS are aligned on the reference genome >10% mapped near (+/- 4 bases) the true insertion site. Without correction, these misaligned sequences not only result in an overestimation of the overall number of IS but in some cases also in the generation of false common insertion sites, worrisome hallmarks of insertional mutagenesis. To mitigate this issue we and others, based on empirical observations, merge sequencing reads mapping within +/- 3 bp into a single IS. Although this adjustment reduces the impact of the "wobbling" around the true ISs, a dedicated method and model is still missing.To further increase the accuracy of genomic positioning of sequencing reads we developed a new bioinformatics framework as post-processing plugin for pipelines that correctly partitions sequencing reads in a given genomic position by considering the relative abundance and distribution of each sequence cluster using local modes and Gaussian scores through an adaptive approach that varies the parameters of the Gaussian curve and proposes different solutions. To chose the best solution, the algorithm first evaluates each solution by exploiting 100 simulations of the input reads and then selects the resulting best solution using the Kolmogorov-Smirnov test. The simulation step is designed to test the mappability of the IS genomic interval and to quantify the impact of the observed nucleotide variations of the reads with respect to the reference genome (PCR artifacts or real genomic differences) that may lead to different mapping results that justify a larger span of the mapped reads surrounding the putative IS. The algorithm returns the list of IS and relative number of reads with the p-value of the best solution.We performed 3 ad-hoc in vitro experiments on a cell clone with 6 known IS in which we measured the precision of IS placement obtaining an average of 100% with our new method whereas <30% using our previous method based on a rigid sliding window approach of 4 bp. We applied our new approach to our clinical trial datasets obtaining improvements in IS genomic placement and overestimation with a reduction of potential false IS of 3% without changing the biological results

674 insertional mutagenesis to identify mechanisms of cetuximab resistance in colorectal cancer

Anti-cancer drugs designed to target specific molecular pathways have shown an excellent therapeutic potential but also very poor long-term durability of tumor responses, mainly due to the outbreak of resistant clones among the residual neoplastic cell population. For that reason, understanding the molecular mechanisms underlying the onset of anti-cancer drug resistance (ACDR) is one of the major goals of clinical research. ACDR has been widely studied by DNA/RNA sequencing of primary human samples and several culprits identified. We have previously developed an approach based on lentiviral vector (LV)-induced insertional mutagenesis that allowed to identify the genes involved in lapatinib and erlotinib resistance on HER2+ human breast cancer cell lines and EGFR+ pancreatic cell line respectively. Here we took advantage of this platform to investigate ACDR genes in colorectal cancer (CRC). Cetuximab, anti-EGFR monoclonal antibody, is used as first line therapy in metastatic CRC, which results in prolonged survival of treated patients. However, nearly all patients relapse due to ACDR. We thus selected CRC cells sensitive to cetuximab deriving either from five microsatellite stable cell lines or from eight Patient Derived Xenografts (PDX), primary human CRC cells implanted subcutaneously into immunodeficient mice (NSG). To induce insertional mutagenesis we generated a luciferase-expressing LV harboring the SFFV enhancer/promoter in the long terminal repeats able to perturb the expression of genes nearby the integration site. As control, we used a non-genotoxic SIN-LV. We set up a collagenase IV-based disaggregation protocol that allows single-cell suspension and a serum-free culture condition to maintain the stemness of in vitro cultured cells. This protocol allowed to efficiently disaggregate and expand CRC cells in vitro as well as reach a LV copy number per cell ranging from 0.25 to 5.6. Luciferase gene expression was stable and allowed live-animal monitoring for up to 30 weeks after transplant. CRC-0069 and -0077 PDXs and NCI-H508 and HDC82 cell lines were transduced ex vivo and kept in vitro and/or transplanted in NSG mice. After in vitro or in vivo expansion of the transduced CRCs cetuximab treatment was applied. After an initial shrinking of the tumor mass in mice we observed ACDR in 3 out of 10 mice transplanted with NCI-H508 cells transduced with SFFV-LV and in none of the controls. Genomic DNA from resistant cells is being used for insertion site (IS) analysis to identify common IS, ACDR gene candidates. IS obtained from SIN-LV groups will be used to filter LV integration biases, whereas IS from SFFV-LV transduced cells but not treated with cetuximab will be used to filter mutations that provide a proliferative advantage unrelated to cetuximab treatment. We will validate the most promising candidates by LV-mediated overexpression and knockdown techniques. This approach could pave the way to perform insertional mutagenesis-based forward genetics studies on primary human samples

529. Lentiviral Vectors with a Reduced Splicing Interference Potential Have a Significantly Improved Safety Profile In Vivo

Genotoxicity assays based on systemic vector injection into newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice has shown that self-inactivating (SIN) lentiviral vector (LV) harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset compared to control mice. Integration site (IS) analysis in vector-induced tumors showed that oncogene activation or tumor suppressor inactivation occurs by mechanisms of aberrant splicing and/or enhancer-mediated overexpression of cellular genes. Although oncogene activation may be reduced by the use of SIN design, moderate cellular promoters and insulator sequences, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. Here, we specifically designed SINLVs harboring sequences complementary to microRNAs (mirT sequence) which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR (mirsT-LTR.LV) or in the vector backbone and outside the gene expression cassette (mirT-LV). In our rationale, the mirT sequences when incorporated in an aberrantly generated mRNA would be selectively degraded through the miRNA pathway. Thus, by taking advantage of our in vivo models, we assessed the genotoxicity of these LVs with mirT sequences. Systemic injection of mirsT-LTR.LV (N=34) and mirT-LV (N=39) in Cdkn2a−/− mice did not cause any significant acceleration in hematopoietic tumor onset compared to un-injected mice (N=37) or mice injected with a SINLV that does not harbor mirT sequences (N=24). Similar results have been obtained after injection of the same vectors in Cdkn2a+/− mice (N=29 mirsT-LTR.LV, N=25 mirT-LV, N=40 un-injected and N=15 injected control mice). To gain additional information on the safety profile of these vectors, we performed IS analysis (N>10,000) in tumor-derived DNA. By this analysis, we previously found that Map3k8 activation by LV insertions was the major mechanism of genotoxicity when prototypical SINLVs were injected into Cdkn2a−/− mice. Now, we found that mice treated with mirsT-LTR.LV and mirT-LV did not show any Map3k8 activating insertions, suggesting that the new vectors are efficient in preventing its activation and confirming their superior safety profile. Furthermore, as expected, Pten was the most frequently targeted gene in tumors derived from Cdkn2a−/− mice injected with the LVs harboring mirT sequences. Pten insertions mainly targeted exons, suggesting the potential inactivation of its transcription unit. Finally, we found that Sfi1 was the major Common Insertion Site (CIS) in Cdkn2a+/− mice injected with LVs harboring mirT sequences. This CIS gene however appears to be the product of an intrinsic bias of LV integration, rather than the result of a selection process. Overall, our studies showed that these new advanced design LVs have a significantly improved safety profile and could represent the vector design of choice in future gene therapy applications

537. New Graph-Based Algorithm for Comprehensive Identification and Tracking Retroviral Integration Sites

Vector integration sites (IS) in hematopoietic stem cell (HSC) gene therapy (GT) applications are stable genetic marks, distinctive for each independent cell clone and its progeny. The characterization of IS allows to identify each cell clone and individually track its fate in different tissues or cell lineages and during time, and is required for assessing the safety and efficacy of the treatment. Bioinformatics pipelines for IS detection used in GT identify the sequence reads mapping in the same genomic position of the reference genome as a single IS but discard those ambiguously mapped in multiple genomic regions. The loss of such significant portion of patients' IS may hide potential malignant events thus reducing the reliability of IS studies. We developed a novel tool that is able to accurately identify IS in any genomic region even if composed by repetitive genomic sequences. Our approach exploits an initial genome free analysis of sequencing reads by creating an undirected graph in which nodes are the input sequences and edges represent valid alignments (over a specific identity threshold) between pairs of nodes. Through the analysis and decomposition of the graph, the method identifies indivisible subgraphs of sequences (clusters), each of them corresponding to an IS. Once extracted the consensus sequence of the clusters and aligned on the reference genome, we collect the alignment results and the annotation labels from RepeatMasker. By combining the set of genomic coordinates and the annotation labels, the method retraces the initial sequence graph, statistically validates the clusters through permutation test and produces the final list of IS. We tested the reliability of our tool on 3 IS datasets generated from simulated sequencing reads with incremental rate of nucleotide variations (0%, 0.25% and 0.5%) and real data from a cell line with known IS and we compared out tool to VISPA and UClust, used for GT studies. In the simulated datasets our tool demonstrated precision and recall ranging 0.85-0.97 and 0.88-0.99 respectively, producing the aggregate F-score ranging 0.86-0.98 which resulted higher than VISPA and UClust. In the experimental case of sequences from LAM-PCR products, our tool and VISPA were able to identify all the 6 known ISs for >98% of the reads produced, while UClust identified only 5 out 6 ISs. We then used our tool to reanalyze the sequencing reads of our GT clinical trial for Metachromatic Leukodystrophy (MLD) completing the hidden portion of IS. The overall number of ISs, sequencing reads and estimated actively re-populating HSCs was increased by an average fold ~1.5 with respect the previously published data obtained through VISPA whereas the diversity index of the population did not change and no aberrant clones in repeats occurred. Our tool addresses and solves important open issues in retroviral IS identification and clonal tracking, allowing the generation of a comprehensive repertoire of IS

Normalization of clonal diversity in gene therapy studies using shape constrained splines

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice

3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences

Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo genotoxicity assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo genotoxicity assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications

27. Aberrant Expression of the Stem Cell microRNA-126 Induces B Cell Malignancy

MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis