Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Genetic determinants of type 1 diabetes in individuals with weak evidence of islet autoimmunity at disease onset.

AIMS/HYPOTHESIS: Islet autoantibodies (AAbs) are detected in >90% of individuals with clinically suspected type 1 diabetes at disease onset. A single AAb, sometimes at low titre, is often detected in some individuals, making their diagnosis uncertain. Type 1 diabetes genetic risk scores (GRS) are a useful tool for discriminating polygenic autoimmune type 1 diabetes from other types of diabetes, particularly the monogenic forms, but testing is not routinely performed in the clinic. Here, we used a type 1 diabetes GRS to screen for monogenic diabetes in individuals with weak evidence of autoimmunity, i.e. with a single AAb at disease onset. METHODS: In a pilot study, we genetically screened 142 individuals with suspected type 1 diabetes, 42 of whom were AAb-negative, 27 of whom had a single AAb (single AAb-positive) and 73 of whom had multiple AAbs (multiple AAb-positive) at disease onset. Next-generation sequencing (NGS) was performed in 41 AAb-negative participants, 26 single AAb-positive participants and 60 multiple AAb-positive participants using an analysis pipeline of more than 200 diabetes-associated genes. RESULTS: The type 1 diabetes GRS was significantly lower in AAb-negative individuals than in those with a single and multiple AAbs. Pathogenetic class 4/5 variants in MODY or monogenic diabetes genes were identified in 15/41 (36.6%) AAb-negative individuals, while class 3 variants of unknown significance were identified in 17/41 (41.5%). Residual C-peptide levels at diagnosis were higher in individuals with mutations compared to those without pathogenetic variants. Class 3 variants of unknown significance were found in 11/26 (42.3%) single AAb-positive individuals, and pathogenetic class 4/5 variants were present in 2/26 (7.7%) single AAb-positive individuals. No pathogenetic class 4/5 variants were identified in multiple AAb-positive individuals, but class 3 variants of unknown significance were identified in 19/60 (31.7%) patients. Several patients across the three groups had more than one class 3 variant. CONCLUSIONS/INTERPRETATION: These findings provide insights into the genetic makeup of patients who show weak evidence of autoimmunity at disease onset. Absence of islet AAbs or the presence of a single AAb together with a low type 1 diabetes GRS may be indicative of a monogenic form of diabetes, and use of NGS may improve the accuracy of diagnosis.

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples.

BACKGROUND: Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. METHODS: To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). RESULTS: Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. CONCLUSIONS: High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

Preexisting Immunity Drives the Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma.

Current treatment for patients with locally advanced esophageal adenocarcinoma (EAC) is neoadjuvant chemotherapy (nCT), alone or combined with radiotherapy, before surgery. However, fewer than 30% of treated patients show a pathologic complete response to nCT, which correlates with increased 5-year survival compared with nonresponders. Understanding the mechanisms of response to nCT is pivotal to better stratify patients and inform more efficacious therapies. Here, we investigated the immune mechanisms involved in nCT response by multidimensional profiling of pretreatment tumor biopsies and blood from 68 patients with EAC (34 prospectively and 34 retrospectively collected), comparing complete responders versus nonresponders to nCT. At the tumor level, complete response to nCT was associated with molecular signatures of immune response and proliferation, increased putative antitumor tissue-resident memory CD39+ CD103+ CD8+ T cells, and reduced immunosuppressive T regulatory cells (Treg) and M2-like macrophages. Systemically, complete responders showed higher frequencies of immunostimulatory CD14+ CD11c+ HLA-DRhigh cells, and reduced programmed cell death ligand 1-positive (PD-L1+) monocytic myeloid-derived suppressor cells, along with high plasma GM-CSF (proinflammatory) and low IL4, CXCL10, C3a, and C5a (suppressive). Plasma proinflammatory and suppressive cytokines correlated directly and inversely, respectively, with the frequency of tumor-infiltrating CD39+ CD103+ CD8+ T cells. These results suggest that preexisting immunity in baseline tumor drives the clinical activity of nCT in locally advanced EAC. Furthermore, it may be possible to stratify patients based on predictive immune signatures, enabling tailored neoadjuvant and/or adjuvant regimens. SIGNIFICANCE: Multidimensional profiling of pretreatment esophageal adenocarcinoma shows patient response to nCT is correlated with active preexisting immunity and indicates molecular pathways of resistance that may be targeted to improve clinical outcomes.

BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY.

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

High surface IgM levels associate with shorter response to ibrutinib and BTK bypass in patients with CLL.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

Radiation-Related Deregulation of TUBB3 and BRCA1/2 and Risk of Secondary Lung Cancer in Women With Breast Cancer.

INTRODUCTION: Breast cancer survivors are at increased risk of developing unrelated primary cancers, particularly lung cancer. Evidence indicates that sex hormones as well as a deregulation of DNA-repair pathways may contribute to lung cancer onset. We investigated whether the hormone status and expression of markers involved in DNA repair (BRCA1/2, ERCC1, and P53R2), synthesis (TS and RRM1), and cell division (TUBB3) might be linked to lung cancer risk. PATIENTS AND METHODS: Thirty-seven breast cancer survivors with unrelated lung cancer and 84 control subjects comprising women with breast cancer (42/84) or lung cancer (42/84) were enrolled. Immunohistochemistry on tumor tissue was performed. Geometric mean ratio was used to assess the association of marker levels with patient groups. RESULTS: Estrogen receptor was expressed in approximately 90% of the breast cancer group but was negative in the majority of the lung cancer group, a result similar to the lung cancer control group. Likewise, ER isoform ß was weakly expressed in the lung cancer group. Protein analysis of breast cancer versus control had a significantly lower expression of BRCA1, P53R2, and TUBB3. Likewise, a BRCA1 reduction was observed in the lung cancer group concomitant with a BRCA2 increase. Furthermore, BRCA2 and TUBB3 increased in ipsilateral lung cancer in women who had previously received radiotherapy for breast cancer. CONCLUSION: The decrease of DNA-repair proteins in breast cancer could make these women more susceptible to therapy-related cancer. The increase of BRCA2 and TUBB3 in lung cancer from patients who previously received radiotherapy for breast cancer might reflect a tissue response to exposure to ionizing radiation.

Prospective Validation of the Italian Alliance Against Cancer Lung Panel in Patients With Advanced Non-Small-Cell Lung Cancer.

BACKGROUND: The deeper knowledge of non-small-cell lung cancer (NSCLC) biology and the discovery of driver molecular alterations have opened the era of precision medicine in lung oncology, thus significantly revolutionizing the diagnostic and therapeutic approach to NSCLC. In Italy, however, molecular assessment remains heterogeneous across the country, and numbers of patients accessing personalized treatments remain relatively low. Nationwide programs have demonstrated that the creation of consortia represent a successful strategy to increase the number of patients with a molecular classification. PATIENTS AND METHODS: The Alliance Against Cancer (ACC), a network of 25 Italian Research Institutes, has developed a targeted sequencing panel for the detection of genomic alterations in 182 genes in patients with a diagnosis of NSCLC (ACC lung panel). One thousand metastatic NSCLC patients will be enrolled onto a prospective trial designed to measure the sensitivity and specificity of the ACC lung panel as a tool for molecular screening compared to standard methods. RESULTS AND CONCLUSION: The ongoing trial is part of a nationwide strategy of ACC to develop infrastructures and improve competences to make the Italian research institutes independent for genomic profiling of cancer patients.

Next Generation Sequencing in Non-Small Cell Lung Cancer: Pitfalls and Opportunities.

Lung cancer remains the first cause of cancer-related deaths worldwide. Thanks to the improvement in the knowledge of the biology of non-small cell lung cancer (NSCLC), patients' survival has significantly improved. A growing number of targetable molecular alterations have been identified. Next-generation sequencing (NGS) has become one of the methodologies entered in clinical practice and was recently recommended by the European society for medical oncology (ESMO) to perform a comprehensive molecular characterization in patients with cancer. The current review provides an overview of the clinical trials that have explored the impact of NGS in patients with cancer, its limits, and advantages.

Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing.

Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management.

ß-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancer.

The limited clinical response observed in high-grade serous ovarian cancer (HG-SOC) with high frequency of TP53 mutations (mutp53) might be related to mutp53-driven oncogenic pathway network. Here we show that ß-arrestin1 (ß-arr1), interacts with YAP, triggering its cytoplasmic-nuclear shuttling. This interaction allows ß-arr1 to recruit mutp53 to the YAP-TEAD transcriptional complex upon activation of endothelin-1 receptors (ET-1R) in patient-derived HG-SOC cells and in cell lines bearing mutp53. In parallel, ß-arr1 mediates the ET-1R-induced Trio/RhoA-dependent YAP nuclear accumulation. In the nucleus, ET-1 through ß-arr1 orchestrates the tethering of YAP and mutp53 to YAP/mutp53 target gene promoters, including EDN1 that ensures persistent signals. Treatment of patient-derived xenografts reveals synergistic antitumoral and antimetastatic effects of the dual ET-1R antagonist macitentan in combination with cisplatinum, shutting-down the ß-arr1-mediated YAP/mutp53 transcriptional programme. Furthermore, ETAR/ß-arr1/YAP gene signature correlates with a worst prognosis in HG-SOC. These findings support effective combinatorial treatment for repurposing the ET-1R antagonists in HG-SOC.

Integrated Somatic and Germline Whole-Exome Sequencing Analysis in Women with Lung Cancer after a Previous Breast Cancer.

Women treated for breast cancer (BC) are at risk of developing secondary tumors, such as lung cancer (LC). Since rare germline variants have been linked to multiple cancer development, we hypothesized that BC survivors might be prone to develop LC as a result of harboring rare variants. Sixty patients with LC with previous BC (the study population; SP) and 53 women with either BC or LC and no secondary cancer (control population; CP) were enrolled. Whole exome sequencing was performed in both tumors and unaffected tissues from 28/60 SP patients, and in germline DNA from 32/53 CP. Candidate genes were validated in the remaining individuals from both populations. We found two main mutational signature profiles: S1 (C>T) in all BCs and 16/28 LCs, and S2 (C>A) which is strongly associated with smoking, in 12/28 LCs. The burden test over rare germline variants in S1-LC vs CP identified 248 genes. Validation confirmed GSN as significantly associated with LC in never-smokers. In conclusion, our data suggest two signatures involved in LC onset in women with previous BC. One of these signatures is linked to smoking. Conversely, regardless of smoking habit, in a subgroup of BC survivors genetic susceptibility may contribute to LC risk.

A CD8α- Subset of CD4+SLAMF7+ Cytotoxic T Cells Is Expanded in Patients With IgG4-Related Disease and Decreases Following Glucocorticoid Treatment.

OBJECTIVE: An unconventional population of CD4+ signaling lymphocytic activation molecule family member 7-positive (SLAMF7+) cytotoxic effector memory T (TEM ) cells (CD4+ cytotoxic T lymphocytes [CTLs]) has been linked causally to IgG4-related disease (IgG4-RD). Glucocorticoids represent the first-line therapeutic approach in patients with IgG4-RD, but their mechanism of action in this specific condition remains unknown. We undertook this study to determine the impact of glucocorticoids on CD4+ CTLs in IgG4-RD. METHODS: Expression of CD8α, granzyme A, perforin, and SLAMF7 within the effector memory compartment of CD45RO+ (TEM ) and CD45RA+ effector memory T (TEMRA ) CD4+ cells was quantified by flow cytometry in 18 patients with active IgG4-RD, both at baseline and after 6 months of glucocorticoid treatment. Eighteen healthy subjects were studied as controls. Next-generation sequencing of the T cell receptor α- and ß-chain gene was performed on circulating CD4+ CTLs from patients with IgG4-RD before and after treatment and in affected tissues. RESULTS: Circulating CD4+ TEM and TEMRA cells were not expanded in IgG4-RD patients compared to healthy controls. CD4+SLAMF7+ TEM cells (but not TEMRA cells) were significantly increased among IgG4-RD patients. Within CD4+SLAMF7+ TEM cells, CD8α- cells but not CD8αlow cells were elevated in IgG4-RD patients. The same dominant clones of CD8α-CD4+SLAMF7+ TEM cells found in peripheral blood were also identified in affected tissue. CD8α- and CD8αlow CD4+SLAMF7+ TEM cells both expressed cytolytic molecules. Clonally expanded CD8α- but not CD8αlow CD4+SLAMF7+ TEM cells decreased following glucocorticoid-induced disease remission. CONCLUSION: A subset of CD8α-CD4+SLAMF7+ cytotoxic TEM cells is oligoclonally expanded in patients with active IgG4-RD. This TEM cell population contracts following glucocorticoid-induced remission. Further characterization of this cell population may provide prognostic information and targets for therapeutic intervention.

Whole exome sequencing of independent lung adenocarcinoma, lung squamous cell carcinoma, and malignant peritoneal mesothelioma: A case report.

The presence of multiple primary tumors (MPT) in a single patient has been identified with an increasing frequency. A critical issue is to establish if the second tumor represents an independent primary cancer or a metastasis. Therefore, the assessment of MPT clonal origin might help understand the disease behavior and improve the management/prognosis of the patient.Herein, we report a 73-year-old male smoker who developed 2 primary lung cancers (adenocarcinoma and squamous cell carcinoma) and a malignant peritoneal mesothelioma (PM).Whole exome sequencing (WES) of the 3 tumors and of germline DNA was performed to determine the clonal origin and identify genetic cancer susceptibility.Both lung cancers were characterized by a high mutational rate with distinct mutational profiles and activation of tumor-specific pathways. Conversely, the PM harbored a relative low number of genetic variants and a novel mutation in the WT1 gene that might be involved in the carcinogenesis of nonasbestos-related mesothelioma. Finally, WES of the germinal DNA displayed several single nucleotide polymorphisms in DNA repair genes likely conferring higher cancer susceptibility.Overall, WES did not disclose any somatic genetic variant shared across the 3 tumors, suggesting their clonal independency; however, the carcinogenic effect of smoke combined with a deficiency in DNA repair genes and the patient advanced age might have been responsible for the MPT development. This case highlights the WES importance to define the clonal origin of MPT and susceptibility to cancer.

REST-Governed Gene Expression Profiling in a Neuronal Cell Model Reveals Novel Direct and Indirect Processes of Repression and Up-Regulation.

The role of REST changes in neurons, including the rapid decrease of its level during differentiation and its fluctuations during many mature functions and diseases, is well established. However, identification of many thousand possible REST-target genes, mostly based on indirect criteria, and demonstration of their operative dependence on the repressor have been established for only a relatively small fraction. In the present study, starting from our recently published work, we have expanded the identification of REST-dependent genes, investigated in two clones of the PC12 line, a recognized neuronal cell model, spontaneously expressing different levels of REST: very low as in neurons and much higher as in most non-neural cells. The molecular, structural and functional differences of the two PC12 clones were shown to depend largely on their different REST level and the ensuing variable expression of some dependent genes. Comprehensive RNA-Seq analyses of the 13,700 genes expressed, validated by parallel RT-PCR and western analyses of mRNAs and encoded proteins, identified in the high-REST clone two groups of almost 900 repressed and up-regulated genes. Repression is often due to direct binding of REST to target genes; up-regulation to indirect mechanism(s) mostly mediated by REST repression of repressive transcription factors. Most, but not all, genes governing neurosecretion, excitability, and receptor channel signaling were repressed in the high REST clone. The genes governing expression of non-channel receptors (G protein-coupled and others), although variably affected, were often up-regulated together with the genes of intracellular kinases, small G proteins, cytoskeleton, cell adhesion, and extracellular matrix proteins. Expression of REST-dependent genes governing functions other than those mentioned so far were also identified. The results obtained by the parallel investigation of the two PC12 clones revealed the complexity of the REST molecular and functional role, deciphering new aspects of its participation in neuronal functions. The new findings could be relevant for further investigation and interpretation of physiological processes typical of neurons. Moreover, they could be employed as tools in the study of neuronal diseases recently shown to depend on REST for their development.

Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model.

Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation.

Exome sequencing and pathway analysis for identification of genetic variability relevant for bronchopulmonary dysplasia (BPD) in preterm newborns: A pilot study.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infancy, affecting preterm children with low birth weight. The disease has a multifactorial aetiology with a significant genetic component; until now published association studies have identified several candidate genes but only few of these data has been replicated. In this pilot study, we approached exome sequencing aimed at identifying non-common variants, which are expected to have a stronger phenotypic effect. MATERIALS AND METHODS: We performed this study on 26 Italian severely affected BPD preterm unrelated newborns, homogeneously selected from a large prospective cohort. We used an Illumina HiSeq 2000 for sequencing. Data analysis was focussed on genes previously associated to BPD susceptibility and to new candidates in related pathways, highlighted by a prioritization analysis performed using ToppGene Suite. RESULTS: By exome sequencing, we identified 3369 novel variants, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family. All of them have been confirmed with Sanger sequencing. CONCLUSIONS: Potential candidate genes have been discovered in this preliminary study; the pathogenic role of identified variants will need to be confirmed with functional and segregation studies and possibly with further methods, able to evaluate the collective influence of rare variants. Moreover, additional candidates will be tested and genetic analysis will be extended to all affected children.