Pharmacokinetics, Bio Distribution and Therapeutic Applications of Recently-Developed siRNA and DNA Repair Genes Recurrence

    

    

    Figure 14: Tissue distribution of 64Cu-DOTA-siRNA delivered by targeted
(Tf) and nontargeted (PEG) NPs. (a) Fused micro-PET/CT images of mice at
1, 10 and 60 min after injection. (b) Blood clearance and tumor localization of
Tf-targeted and non-targeted siRNA NPs.
Reprinted with permission from Reference [114]. Copyright (2007) National
Academy of Sciences, USA.
    

Pharmacological effects of siRNA therapeutics

Due to the favorable BD of siRNA delivery systems, the liver and lung are among the primary targets of siRNA therapeutics. This section provides a brief overview of recent preclinical studies on pharmacological effects of siRNA therapeutics in liver and lung diseases.

Liver diseases

HCC: Initial efforts to treat HCC with siRNA involved free siRNA injection. siRNA targeting VEGF expression in hepatoma and endothelial cells with no specific carrier system was tested in a murine orthotopic hepatoma model [61]. Animals were injected with 200 μg/kg of siRNAVEGF intraperitoneally, every 2 days for 14 days starting from24 h prior to tumor cell inoculation. When determined terminally, tumor burden was reduced by 63% [61]. The antitumor efficacy was attributed to the inhibition of tumor angiogenesis, corresponding to intratumoral microvessel density [61]. Similarly, siRNA targeting antiapoptotic protein heme oxygenase 1 (HO-1) was intraperitoneally injected to immune-competent mice bearing HO-1+ Hepa129 tumors in the liver and reduced tumor growth by 50% with no damages in normal organs [128]. The authors [129] attributed this effect to proapoptotic and possibly antiangiogenic activity of siHO-1 [128].

For improving siRNA delivery to the liver andHCC, NPs based on cationic lipid such as 1,2-dioleyloxy-3-trimethylammonium propane (DOTAP) are widely used as a carrier of siRNA. Schmitz et al. made a complex of VEGF-targeting siRNA with DOTAP and injected the complex intraperitoneally multiple times to mice with pre-existing liver fibrosis as well as orthotopic HCC (Hepa129 cells injected to the left liver lobe) [130]. The VEGF-siRNA/DOTAP complexes resulted in significant reduction in the number of HCC satellites in the liver (82% mice had < 30 satellites), whereas free VEGF-siRNA and control siRNA had a minimal effect (22.1% and 12.5% mice with < 30 satellites, respectively). DOTAP helped VEGFsiRNA enter HCC satellites and silence VEGF expression in hepatic tissues. However, the effect of the internalized VEGFsiRNA-DOTAP complex on tumors was more likely through immune activation via interferon-1 upregulation than VEGF silencing [130]. In fact, intratumoral VEGF expression was not reduced by VEGF-siRNA/ DOTAP complexes but rather increased [130]. More recently, Shen et al. screened several lipid NPs for optimal siRNA delivery to HCC and identified a combination of lipids that showed significant anti-tumor effect with cancer-targeting siRNAs [131]. An interesting note in this study is that good in vitro transfection efficiency did not necessarily translate to good in vivo silencing effect in orthotopic HCC tumors, which indicates the significance of circulation stability of gene–carrier complexes [131].

To further improve cell specificity of siRNA delivery, cellinteractive ligands have been added to the carrier. For example, a model siRNA was encapsulated in PEGylated DOTAP-based immunoliposomes decorated with anti-EGFR Fab’ [132]. A BD study in an orthotopic mouse model of HCC showed that the immunoliposomes accumulated in the liver and were taken up by HCC cells to greater extents than non-targeted liposomes, achieving higher silencing effect on the model luciferase gene expression [132]. This system was later used for the co-delivery of doxorubicin and siRNA targeting RRM2, a gene important for DNA synthesis and repair and highly expressed in HCC [126]. The siRNA and drug delivered with immunoliposomes showed 50–60% ID/g in the HCC tumors, compared to 20-30% ID/g of non-targeted liposomes, 8 h after injection. Accordingly, animals treated with siRNA-RRM2 and doxorubicin in immunoliposomes showed the most significant retardation of tumor growth, compared to those receiving each treatment or dual loads in non-targeted liposomes [126].

Liver infections: Due to the preferable BD in the liver, hepatitis infections like HBV and HCV were one of the earliest targets of siRNA therapeutics. For example, siRNA targeting the S region of HBV RNA was delivered with SNALPs based on a mixture of cationic and fusogenic lipids [133,134]. Here, 2'-OH of siRNA was chemically modified to increase the resistance against nucleases [133]. IVinjected SNALP particles were accumulated predominantly in the liver (28%) with minimal accumulation in other organs (8.2% in spleen and 0.3% in the lung) by 24 h after injection [133]. Reflecting the high liver accumulation, siRNA delivered with SNALPs achieved a significant reduction of the level of HBV DNA copies and HBsAg proteins for 7 days after dosing [133]. In another case, siRNAs targeting HBV transcript sites 1407 or 1794 were delivered with lipid assemblies based on three different lipids, including one conjugated to PEG2000 by oxime linkages, which could be cleaved at pH lower than 5.5 [78]. A BD study with the radiolabeled assemblies found that regardless of the PEG concentration in the formulation, most of the dose (>50%) was detected in the liver after 1 h post-injection, and other organs such as spleen, kidney, blood and lung showedminimal accumulation of the particles. Accordingly, HBV transgenic mice receiving the siRNA-lipid assemblies showed significant reduction in HBV replication, indicated by the reduced levels of HBV mRNA and HBeAg proteins in the liver and HBsAg in serum, equivalent to daily intraperitoneal treatment of Lamivudine, a licensed drug for HBV treatment [78]. Later, this group introduced a new cationic lipid DODAG (N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycylamide) and achieved a similarly effective knock-down of HBV infection [135].

siRNA targeting 5'-UTR of HCV genome was encapsulated in liposomes composed of a cationic lipid, Phosphatidylcholine (PC) and lactosylated phosphatidylethanol, which targeted the asialoglycoprotein receptor on hepatocytes [86]. A BD study with a fluorescently labeled (Alexa-568) siRNA in BALB/c mice showed preferential accumulation of siRNA in the liver in 30 min after injection with the lactosylated liposomes, whereas those delivered with non-lactosylated liposomes showed equally strong signals in the spleen and liver [86]. In a pharmacodynamics study with an HCV transgenic mouse model, siRNA targeting 325-344 nucleotides of HCV genome, delivered with the lactosylated liposomes, efficiently suppressed HCV protein expression in a dose dependent manner 48 h after a single treatment [86]. Similarly, siRNA was incorporated in cationic liposomes (‘DTC-apo’) composed of DOTAP and cholesterol decorated with apolipoprotein A-1, the protein component of highdensity lipoprotein (HDL) targeting hepatocytes. ABD study using fluorescently labeled 21-mer dsDNA encapsulated in DTC-apo showed relatively high fluorescence intensity in the liver compared to other organs like kidney, lung, and spleen [89]. Without the apolipoprotein ligand, fluorescence was preferentially seen in the lung and spleen [89]. In vivo silencing effect was tested in a HCV mouse model created with hydrodynamic injection of plasmid DNA expressing viral structural proteins. HCV-specific siRNA/DTC-apo complex inhibited viral gene expression by 65% in the liver at 2 mg/ kg siRNA on day 2 post administration. The extent and duration of siRNA were further improved by 2'-O-methylmodification of siRNA [89]. ApolipoproteinA-1was later replaced with a recombinant form of apolipoprotein (rhApo) derived from Escherichia coli to avoid the safety concern (e.g., pathogen contamination) related to the origin (plasma) [136]. In addition to siRNA, synthetic shRNA targeting the IRES of the HCV genome was incorporated in a mixture of lipids to form lipid NPs, showing long lasting potent efficacy against HCV in mice with minimal toxicity [87].

More recently, it was demonstrated that simple co-injection of a hepatocyte-targeted peptide could help deliver siRNA targeting conserved HBV sequences to the liver [77]. The peptide consisted of N-acetylgalactosamine (NAG), a ligand with high affinity for asialoglycoprotein receptors highly expressed on the hepatocytes, and melittin-like peptide (MLP), an endosomolytic agent to facilitate endolysosomal escape of siRNA [77]. A co-injection of NAGMLP with liver-tropic cholesterol-conjugated siRNA increased the hepatic gene silencing effect in a dose-dependent manner, resulting in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect in mouse models of HBV infection. This formulation is unique in that it does not require preformulation of the complex like typical siRNA delivery systems, thus reducing the complexity in large-scale manufacturing. It has recently entered phase IIa clinical trial for the treatment of chronic HBV infection (NCT02065336) [137].

As one of the primary sites for Ebola virus replication, the liver also serves as a valid target for siRNA therapy of Ebola virus infection. siRNA targeting individual regions of Zaire Ebola virus (ZEBOV) L gene was delivered with SNALPs [92]. Despite the evidence suggesting premature release of siRNA in circulation (differential BD of labeled SNALPs and siRNA), siRNA/SNALPs provided post-exposure protection to guinea pig challenged with a lethal Ebola virus. Animals challenged with ZEBOV received siRNA treatment at 1, 24, 48, 72, 96, 120, and 144 h after the infection. On day 30, 100% survival rate (n=5) was observed in the group treated with the siRNA/SNALP formulation, while the control group treated with scrambled siRNA/ SNALPs presented 20% survival rate. Consistently, the plague titer analysis on day 7 showed a significantly reduced blood level in animals treated with siRNA/SNALPs compared to the control group. Later this group encapsulated a cocktail of three siRNAs that each targeted L protein, VP24 and VP35 genes in ZEBOV in SNALPs and showed complete post-exposure protection against ZEBOV in monkeys [91]. Tekmira Pharmaceuticals commenced a Phase I clinical trial for the cocktail siRNA–SNALP formulation (TKM-100802) in Jan 2014 (NCT02041715) [138]. Two patients transported from West Africa to the United States during the 2014 outbreak of Ebola virus disease were treated with TKM-100802 and other supportive measures and recovered without serious long-term sequelae, although the actual role the siRNA formulation played in the recovery of these patients remains to be investigated [139].

Respiratory diseases: For systemic delivery of siRNA to the lung, cationic polymers such as PEI are frequently used as carriers [140]. In an early study by Klibanov et al., deacylated PEI was used as a carrier of siRNA to the lung [99]. Here, deacylation increased the number of protonatable nitrogens of PEI, thereby facilitating complexation with siRNA and transfection of target cells. Luc-siRNA-deacylated PEI complexes were administered to mice transfected with luciferaseencoding DNA by retroobrital injection, and the luciferase activities in different tissues were measured at 24 h after the treatment. The Luc-siRNA/polymer complexes showed 77 or 93% suppression of the gene expression in the lung, depending on the molecular weight of the polymer. Consistently, siRNA targeting influenza viral genes, as complexed with deacylated PEI, led to significant reduction in the virus titer in the lung of influenza-infected mice 24 h after injection [99]. More recently, Bolcato-Bellemin et al. used PEI as a carrier of siRNAwith sticky overhangs (ssiRNA) targeting cyclin B1 or survivin to suppress tumor progression in a lung metastasis model [141]. Here, ssiRNA formed a larger linear nucleic acid structure via hybridization of the complementary overhangs andmade a more stable complex with PEI, which survived better than a classic siRNA/PEI complex in circulation [142].

Cationic liposomes tend to accumulate in the liver and hence are popular in the delivery to the liver, but they are also used for lung delivery. McMillan et al. used cationic liposomes containing DOTAP and DOPE as a carrier of siRNA targeting lung endothelium [143]. A BD study with the liposomes labeled with a non-covalently incorporated fluorescent dye found the highest fluorescence intensity was found in the liver, followed by lung and spleen.The liposomes transfected various cell populations in the lung, including CD146+ endothelial cells and CD326+ epithelial cells. Accordingly, mice treated with lamin A/C-specific siRNA-liposomes (2mg/kg siRNA) showed 80% knockdown of lamin A/C in the lung at 48 h after a single IV injection, with the majority silencing occurring in the endothelial and epithelial cells [143]. A new choice of cationic lipids and combinations improved lung-specific BD of liposomes [144]. Kaufmann et al. used “DACC” liposomes containing AtuFECT01 (β-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-Noleyl-amide trihydrochloride) as a cationic lipid for systemic administration of siRNA. When combined with cholesterol and mPEG2000-DSPE in optimal ratios, DACC liposomes delivered the most siRNA to the lung tissues at 1 h after systemic application, followed by the spleen and liver. On the other hand, other liposomes containingAtuFECT01 and different colipids and/or PEG-lipids showed typical BD pattern of cationic liposomes (high liver and spleen distribution) [144]. In accordance with the BD profile, DACC lipoplexes with siRNA specific for Tie-2 gene showed over 80% reduction in Tie-2 mRNA in lung tissue of mice but no significant Tie-2 knockdown in liver, kidney and heart tissues after 3 daily injections of 2.8 mg siRNA/ kg [144]. DACC lipoplexes also helped deliver siRNAs targeting other genes in the lung vasculature including CD31, responsible for multiple processes of tumorigenesis, increasing the survival rate in an experimental lung metastasis mouse model [144]. Geick et al. also found BD favorable for lung delivery of siRNA with a lipopolyamine called staramine [145]. siRNA level in each tissue wasmeasured by the stem-loop qRT PCR over 96 h post-intravenous injection. Liver accumulation was dominant immediately after the injection, but lung accumulation occurred over time, exceeding the levels in the liver, spleen, and kidney in 24 h. Consistently, siRNA/staramine complexes showed ~60% reduction of target mRNA in the lung over 96 h after a single administration, whereas the level of target mRNA in the liver was not significantly different from the one treated with control siRNA [145]. The siRNA retention in the lung was attributed to the affinity of nanocomplexes for the endothelial lining in the capillary bed and relatively low exposure to phagocytic cells [145].

Other types of siRNA carriers to the lung include hybrids of lipid and polymer or lipid and inorganic components. Polymeric NPs made of conjugates of low molecular weight polycation (PEI) and lipids were used for delivery of siRNA to lung endothelial cells [146]. Unlike lipid-based carriers, the lipid–polymer hybrid NPs achieved the gene silencing effect at a dose that did not affect hepatocytes or immune cells. The biological effects of siRNAs delivered with the NPs were confirmed in the lung, as an inducer of an emphysema-like phenotype (by silencing VEGFR-2 gene) or a suppressor of primary and metastatic Lewis lung carcinoma (by silencing VEGFR-1 and Dll4) [146].

In addition to systemic administration, siRNA has been directly delivered to the lung as an aerosol (intratracheal or intranasal administration in experimental studies). One of the advantages of airway delivery is the potential to achieve high local concentration of siRNA in the lung without going through blood circulation, which can compromise the stability of the siRNA/carrier complexes or siRNA itself and subject them to distribution in other MPS organs. Minko et al. administered siRNA/cationic liposome complexes via intravenous injection or intratracheal instillation to mice bearing tumors in the lung to compare the BD [147]. At 24 h postadministration, both the carrier and siRNA showed substantially high peak concentrations and long retentions in the lung compared to other organs after intratracheal administration. On the other hand, IV injection resulted in broad distribution in other organs with relatively low levels of liposomes and siRNA in the lung [147]. Kissel et al. also delivered siRNA with PEI or PEG-grafted PEI (PEG-PEI) as carriers via intratracheal instillation and observed the BD of siRNA and the polymer by single photon emission computed tomography (SPECT) and radioactivity counting of organs [148]. Both siRNA and PEG-PEI were detectable in the lung at 48 h after instillation. Without PEG, siRNA/PEI complexes showed differential lung accumulation of each component: negligible level of siRNA and high level of PEI (even higher than that of PEG-PEI), which suggested dissociation of the complex in the lung and preferential association of PEI with mucus and cell membranes [148]. The locally delivered EGFP-specific siRNA/PEG-PEI complexes reached bronchial cells and alveolar cells and knocked down the EGFP expression in the lung by 42% after 5 days post-instillation [148]. Airway administration is potentially a promising way of delivering siRNA to the lung; however, studies to date suggest remaining challenges in airway delivery of siRNA, including the destructive interaction with anionic components in the lung [148] and local irritation that can lead to inflammatory consequences [149].

Potential toxicity of siRNA therapeutics: siRNA with suboptimal potency can result in unintended side effects such as downregulation of off-target genes or activation of innate immune responses [150]. Efforts to mitigate these off-target effects include chemical modification of siRNA, redesign of the sequence, and the use of siRNA cocktails [150-152]. Toxicity may also come from the delivery systems. For example, intravenously injected lipoplexes have shown to induce the production of pro-inflammatory cytokines with signs of hepatocyte injury [153,154]. These pro-inflammatory effects were initially attributed to the polynucleotide part of the complex rather than the carrier [153,154]; however, later studies have reported intrinsic pro-inflammatory properties of some cationic lipids [155- 157]. PEI, the most commonly used polymeric gene carrier, shows molecular weight, structure, and dose-dependent toxicity [158]. This challenge is currently tackled by developing degradable polycations, which can be degraded into less toxic small molecules [159].

DNA Recurrence-associated genes

It is important to identify systemically recurrence-associated genes using The Cancer Genome Atlas RNA sequencing database [160]. Recurrence of papillary thyroid cancer is not uncommon, but incorporating clinicopathologic parameters to predict recurrence is suboptimal. It has been found that [160] 1,807 genes were associated with recurrence-free survival. There were 676 genes of which high expression was associated with a greater risk of recurrence. These genes were enriched in pathways involved in cell cycle regulation and DNA repair. Among 1,131 genes of which low expression was associated with recurrence, Kyoto Encyclopedia of Genes and Genomesannotated functions were metabolism, calcium signaling, glycan biosynthesis, and the Notch signaling pathway. Canonical pathways identified by Ingenuity Pathway Analysis included RXR function, nitric oxide signaling, interleukin-8 signaling, and nutrient sensing. In addition, low expression of the majority of thyroid differentiation genes was associated with a significantly less recurrence-free survival.

The incidence of thyroid cancer, particularly papillary thyroid cancer, has been increasing rapidly in recent decades [161]. Papillary thyroid cancer tends to be biologically indolent and has an excellent prognosis. Nonetheless, more than one-quarter of patients may experience disease recurrence during long-term follow-up [162]. Several schemes, including clarification of the staging classification of the American Joint Committee on Cancer, have been developed to predict risk for death but not the risk for recurrence.

In 2009, the American Thyroid Association (ATA) first proposed a 3-level, initial risk-stratification system to predict disease recurrence and/or persistence [163]; however, the proportion of variance explained by the ATA system for risk of recurrence is suboptimal, ranging from 19% to 34% [164]. Although a new, dynamic, riskstratification system has been developed, the information required for stratification is available mainly during the follow-up period. This situation indicates a need for early identification of additional factors associated with disease recurrence.

Genetic and molecular approaches are being used increasingly for recognizing important biologic processes in oncology. The most frequent genetic alteration seen in papillary thyroid cancer is a point mutation at codon 600 of the BRAF gene (BRAFV600E) [165]. It remains debatable whether this information concerning this mutation added predictive value for disease-specific mortality or risk of recurrence beyond the current clinicopathologic information collected for tumor staging [166].

In recent years, the prognostic importance of the TERT promoter mutations in thyroid cancer has attracted much attention; nonetheless, the frequency of the TERT promoter mutations in papillary thyroid cancer is considerably less than that in poorly differentiated or anaplastic cancer [167]. Given the biologic complexity of cancer, it is unlikely to find a single target accounting for disease recurrence. A systemic approach is required to identify a gene expression signature associated with recurrent disease.

The Cancer Genome Atlas (TCGA) project is meant to produce a comprehensive atlas of cancer genomes to accelerate our understanding of the molecular basis of cancer and improve cancer prevention, detection, and therapy. The TCGA study of papillary thyroid cancer analyzed a large cohort of tumor tissue and matched normal tissues across multiple sequencing platforms [168]; however, the follow-up data were not available at the time of initial TCGA publication. In this study [160], it has been e employed a systematic approach for characterizing recurrence-associated genes and their relevant functional networks using the updated TCGA data.

Functional enrichment analyses: Recurrence-associated genes were subjected to functional enrichment analyses [160], which were conducted using 3 software packages separately: Web-based Gene Set Analysis Toolkit (WebGestalt), [169] Database for Annotation, Visualization, and Integration Discovery (DAVID), [170] and Ingenuity Pathway Analysis (Qiagen, Redwood City, CA) [171]. WebGestalt and DAVID analyses were focused mainly on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation database [172,173]. In WebGestalt, the minimum number of genes for enrichment was set at 5, and the significance analysis was performed using the hypergeometric test with the significance level set at P = .01. In DAVID, the parameters were set to their default values.

Using the TCGA thyroid cancer database, 504 patients with data for both clinical and gene expression were analyzed. Pathologic stages I, II, III, and IV comprised 56%, 10%, 22%, and 11%, respectively. The median follow-up duration was 29.9 months (mean, 37.7 ± 32.1; range, 0.2–180.8 months). During follow-up, 16 (3%) patients were deceased at 39.0 ± 24.5 months. Recurrence was observed in 47 (9%) patients. The median time from initial treatment to recurrence was 16.1 months (mean, 19.6 ± 14.7; range, 0.2–59.5 months). As expected, an advanced pathologic stage was associated with a greater likelihood of recurrence [160].

Among 20,502 genes analyzed, the distribution of median RSEM values was significantly skewed (median, 187.0; interquartile range, 4.1–800.0). To avoid variation caused by low-level expressions, genes with a median RSEM value of less than 4.1 were excluded from analysis. A total of 15,374 (75%) genes were applicable to the analysis of the difference in RFS between high- and lowexpression groups defined by median splits. A study flowchart is detailed in Figure 15.

Figure 15: Study flowchart. RFS, Recurrence-free survival; RSEM, RNASeq by expectation maximization; TCGA, The Cancer Genome Atlas; THCA, thyroid cancer.

    

    

    Figure 15: Study flowchart. RFS, Recurrence-free survival; RSEM, RNASeq
by expectation maximization; TCGA, The Cancer Genome Atlas; THCA,
thyroid cancer.
    

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There were 676 genes of which high expression was associated with a significantly lesser RFS [160]. The overlapping KEGGenriched pathways were determined at the significance levels of P < .01 in WebGestalt and P < 0.1 in DAVID, respectively. Of the 10 enriched pathways, most were biologic processes involved in cell cycle regulation and DNA repair [160]. The Kaplan-Meier estimates of RFS of several representative genes are shown in Figure 16.

Figure 16: Kaplan-Meier recurrence-free survival of patients with papillary thyroid cancer (n = 504) dichotomized according to genes of which high expression was associated with recurrence.

    

    

    Figure 16: Kaplan-Meier recurrence-free survival of patients with papillary thyroid cancer (n = 504) dichotomized according to genes of which high expression was
associated with recurrence.
    

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Good concordance was observed between the enriched functional pathways identified by the KEGG module and Ingenuity Pathway Analysis (Figure 17A). The top tox lists of Ingenuity Pathway Analysis were “Cell Cycle: G2/M DNA Damage Checkpoint Regulation,” “Increases Liver Hyperplasia/Hyperproliferation,” “Cell Cycle: G1/S Checkpoint Regulation,” “RAR Activation,” and “p53 Signaling.” Consistent with a recent report [174] , it has been found that expression of BRAF mRNA was not associated with RFS (logrank P = 0.76).

Figure 17: Ingenuity Pathway Analysis of genes of which high (A) and low (B) expression was associated with recurrence in papillary thyroid cancer.

    

    

    Figure 17: Ingenuity Pathway Analysis of genes of which high (A) and low (B) expression was associated with recurrence in papillary thyroid cancer.
    

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In addition, we found 1,131 genes for which low expression was associated with a significantly less RFS. The complete list of these genes is reported in [160]. The 9 significantly enriched KEGG pathways identified by WebGestalt and DAVID were related to metabolism, calcium signaling, glycan biosynthesis, and the Notch signaling pathway [160]. The results appeared partially consistent with canonical pathways identified by Ingenuity Pathway Analysis (Figure 17B). The top tox lists were “LPS/IL-1 Mediated Inhibition of RXR Function,” “Cardiac Hypertrophy,” “Xenobiotic Metabolism Signaling,” “Increases Glomerular Injury,” and “NRF2-mediated Oxidative Stress Response”.

Initial TCGA global analysis proposed that the differentiation status of thyroid cancer can be determined by a set of genes involved in thyroid metabolism and function and were designated the Thyroid Differentiation Score (TDS) [168]. The TDS comprises expression data of 13 mRNAs and 3 miRNAs. Interestingly, we found that the expression of 7 TDS genes was in association with RFS (Figure 18). Low expression of these TDS genes was associated typically with a decrease in RFS. The only exception was PVRL4, high expression of which had a greater risk of recurrence. Low expression of TSHR, which encodes thyroid-stimulating hormone receptor, was also associated with a significantly less RFS. It has been shown that [160] the results of the remaining TDS genes. RFS was not associated with other genes participating in thyroid development, such as NKX2-1 (TTF1, logrank P = 0.74), FOXE1 (TTF2, log-rank P = 0.75), and PAX8 (log-rank P = 0.19).

Figure 18: Kaplan-Meier recurrence-free survival of patients with papillary thyroid cancer (n = 504) dichotomized according to genes in association with thyroid differentiation. The sodium iodide symporter (NIS) is encoded by the solute carrier family 5 member 5 (SLC5A5).

    

    

    Figure 18: Kaplan-Meier recurrence-free survival of patients with papillary thyroid cancer (n = 504) dichotomized according to genes in association with thyroid
differentiation. The sodium iodide symporter (NIS) is encoded by the solute carrier family 5 member 5 (SLC5A5).
    

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Genomic global analysis of the TCGA

Initial genomic global analysis of the TCGA of papillary thyroid cancer revealed that papillary thyroid cancers can be separated into 2 main groups, BRAFV600E-like and RAS-like [168]. The classifier design was based on the BRAFV600E-RAS score, which highly correlated with the TDS, although the 2 scores were derived independently and have no genes in common. BRAFV600E-like papillary cancers are enriched for the classic and tallcell histology, are less differentiated, and have an increased ERK output or overactivation of the mitogen-activated protein kinase (MAPK) pathway. Conversely, RAS-like cancers are characterized by follicular-variant histology, are associated with younger patients, and have a lesser expression of immune response genes. The 2 groups are fundamentally different at all genomic levels.

Using the ATA risk of recurrence as a surrogate outcome, the initial analysis found that the risk of recurrence was associated with mutation density and the TDS. The TDS only weakly correlated with tumor purity, indicating that the TDS was not influenced strongly by tumor stromal content or lymphocyte infiltration [168]. The analysis on real events during follow-up are in concordance with the results [160], showing that low expression of thyroid differentiation genes carried a greater risk of recurrence.

In a previous report, histopathologic characterization of radioactive iodine (RAI)–refractory disease suggested that dedifferentiation is not uncommon [175]. Dedifferentiation likely dampens the responses to RAI therapy. At present, patients with an RAI-refractory disease have limited alternative options for treatment and a decreased overall survival, partly because thyroid cancer is poorly responsive to cytotoxic chemotherapy [176].

Cancer is driven by changes in patterns of gene expression due to the accumulation of mutations or epigenetic modifications. The TCGA data support previous observations that RAI-refractory lesions have a greater frequency of BRAFV600E [177,178]. Mutations leading to constitutive activation of the MAPK signaling pathway inhibit the expression of proteins involved in the biosynthesis of thyroid hormones, including sodiumiodide symporter SLC5A5 (NIS) [179]. Of note, among the thyroid differentiation genes, it has been found that downregulation of NIS was the strongest predictor of a decreased RFS (Figure 18, logrank P = .0003). Nuclear receptor ligands and epigenetic modifiers have been used to promote tumor redifferentiation, [180] while conventional redifferentiation agents, such as retinoic acid, had no clinically important benefits [181].

A plausible explanation is that pathways involving retinoic acid receptors and/or retinoid: X receptors are also downregulated in these neoplasms. In these analyses, low expression of retinoic acid receptor beta (RARB) was associated with a significantly lesser RFS (log-rank P = .03). Consistent with this observation, recent studies demonstrated that RARB expression was downregulated in papillary thyroid cancer and RARB silencing increased cell viability, decreased expression of NIS, and suppressed iodine uptake in thyroid cancer cells [182,183]. A more promising strategy may be targeting the MAPK signaling pathway. Accumulating evidence suggests that MAPK inhibition may provide better results in terms of iodine uptake and tumor response [184-186]. Further research is necessary to provide a more personalized approach, tailored to specific molecular alterations in individual patient tumors.

A novel finding in the present study [160] is that high expression of cell cycle–regulating and DNA repair genes was associated with recurrence. Alterations in cell cycle–regulating genes promote transition through the cell cycle and ultimately drive tumor proliferation. Although prior studies suggest that Ki-67/MIB-1 stain positivity confers a greater risk of recurrence, [187] differentiated thyroid cancer has long been considered as a quiescent, chemotherapyresistant neoplasm. It is arguable whether modern drugs offer better effectiveness with fewer adverse events [188].

The findings [160] do not imply that cytotoxic agents possess high therapeutic potential in papillary thyroid cancer. Transcriptome analysis across 12 TCGA cancer types indicates that papillary thyroid cancer is poorly overlapped with other cancers in terms of differentially expressed genes [189]. Instead, it has been speculated that a proliferation signature may be used as a prognostic marker.

DNA repair machinery maintains a balance between genomic integrity and cell death. Previous work mainly focused on the association between polymorphisms of DNA repair genes and the risk of developing thyroid cancer [190]. Upregulation of DNA repair genes may render tumor cells less vulnerable to DNA damage and more resistant to treatment. It has been found that high expression of DNA repair genes was associated with an increased risk of recurrence. It may reflect genomic instability of more aggressive cancer; an alternative explanation is that tumors equipped with better repair capacity would more likely survive from RAI therapy.

The TCGA analysis showed that 2 significantly mutated genes related to DNA repair (PPM1D and CHEK2) occurred concomitantly with driver mutations in the MAPK pathway [168]. Interestingly, MAPK inhibition may result in radiosensitization by suppressing DNA repair pathways [191]. Combination or sequential treatments with RAI therapy and inhibition of the DNA-damage response may represent a new concept for the management of papillary thyroid cancer.

Limitations of this study [160] include the relatively short followup periods and not controlling for histologic variants or tumor stage of the papillary thyroid cancers studied. Additionally, in the present study, patients were divided into dichotomous groups for each gene analyzed. The underlying pathology is more likely to be “continuous” rather than categorical, and such groupings may not be meaningful biologically [192].

A recent analysis of the TCGA data by Stanford University used clinicopathologic characteristics to define good and poor prognosis [193]. The authors [160] reported individual potential targets and proposed a Prediction Analysis of Microarrays classifier. This study aimed at identifying recurrence-associated genes instead of developing a validated prognostic index. It has been hypothesized that using pathway analysis would provide a better mechanistic understanding of the biologic processes involved in tumor recurrence.

Conclusions

We discussed several siRNA delivery approaches focusing on their contributions to the control of PK and BD of siRNA. Solid tumors are obviously one of the most anticipated targets, and many studies show PK/BD profiles favorable for siRNA delivery to tumors. Despite the optimism prevalent in the literature, one may recognize that most clinical translations are made in the applications targeting the liver and the lung, where the majority of siRNA/NP systems naturally accumulate. This indicates that systemic delivery of siRNA in a therapeutic dose to tissues other than MPS organs remains suboptimal. Given the discrepancy, some of the current experimental practices may be worth reconsideration. First, many studies rely on labeling siRNA for PK and BD studies. As mentioned earlier, these approaches are valid only when the labeled components represent the full-length, bioactive siRNA; otherwise, inactive catabolites retaining the label may be mistaken for siRNA in target tissues. In addition, one should also consider that structural changes due to labeling might alter the PK/BD profiles of siRNA. To avoid overestimation of siRNA in PK/BD studies, it is desirable to complement with hybridizationbased methods that can detect full-length siRNA, such as Northern blot analysis, hybridization-ligation assay, or PCR-based assay. Second, BD of siRNA is often presented as % of injected dose per unit mass of tissue at the final time point of the study. While this presentation offers a useful means to compare the relative concentrations of siRNA in different tissues, it rarely reports the amount of siRNA collected in the feces and urine, making it difficult to judge the fraction of siRNA that was immediately excreted. A caveat of this practice is that relative siRNA distribution in selected tissues can mislead to an overrated optimism for target-specific siRNA delivery, when in reality the absolute amount of siRNA in target tissue may be only a small fraction of the injected dose. Such misinterpretation may be prevented by considering the concentration in each tissue in addition to the relative quantity. Finally, it is not unusual to see studies tracking carriers in lieu of siRNA in PK/BD studies. This approach may be valid when the siRNA/carrier complex remains stable throughout the study, which, however, should not be taken for granted without proof. It is possible that siRNA/carrier complexes disassemble prematurely during circulation but still show favorable pharmacological effects due to other biological events related to the carrier itself. In order to identify actual contribution of carriers to the siRNA delivery, it is desirable to track siRNA and carrier separately through distinct labeling and/or detection techniques.

In conclusion, we identified a set of recurrence-associated genes in papillary thyroid cancer. Pathway analyses indicate that upregulation of cell cycle–regulating genes and DNA repair genes and downregulation of thyroid differentiation genes carried a high risk of recurrence. Further research [194-197] will be required to determine whether pharmacologic measures that target these pathways improve clinical outcomes.

References

1. A Fire, S Xu, MK Montgomery, SA Kostas, SE Driver, CC Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature. 1998; 391: 806-811.
2. SM Elbashir, J Harborth, W Lendeckel, A Yalcin, K Weber, T Tuschl. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature. 2001; 411: 494-498.
3. A Dose Escalation Trial of an Intravitreal Injection of Sirna-027 in Patients With Subfoveal Choroidal Neovascularization (CNV) Secondary to Age- Related Macular Degeneration (AMD).
4. D Haussecker. Current issues of RNAi therapeutics delivery and development, J. Control. Release. 2014; 195: 49-54.
5. S Seth, R Johns, MV Templin. Delivery and biodistribution of siRNA for cancer therapy: challenges and future prospects, Ther. Deliv. 2012; 3: 245- 261.
6. R Kanasty, JR Dorkin, A Vegas, D Anderson. Delivery materials for siRNA therapeutics, Nat. Mater. 2013; 12: 967-977.
7. Study of ARC-520 in Patients with Chronic Hepatitis B Virus.
8. A Guzman-Aranguez, P Loma, J Pintor. Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy, Br. J. Pharmacol. 2013; 170: 730-747.
9. SJ Reich, J Fosnot, A Kuroki, W Tang, X Yang, AM Maguire, et al. Tolentino, Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model, Mol. Vis. 2003; 9: 210-216.
10. M Murata, T Takanami, S Shimizu, Y Kubota, S Horiuchi, W Habano, et al. Inhibition of ocular angiogenesis by diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF), Curr. Eye Res. 2006; 31: 171-180.
11. B Kim, Q Tang, PSBiswas, J Xu, RM Schiffelers, FY Xie. et al. Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes, Am. J. Pathol. 2004; 165: 2177-2185.
12. PK Kaiser, RC Symons, SM Shah, EJ Quinlan, H Tabandeh, DV Do, et al. Sirna-027 Study, RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027, Am J. Ophthalmol. 2010; 150: 33-39 e32.
13. RN Frank, Diabetic retinopathy. N. Engl. J. Med. 2004; 350: 48-58.
14. AP Adamis, JW Miller, MT Bernal, DJ D’Amico, J Folkman, TK Yeo, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy, Am J. Ophthalmol. 1994; 118: 445.450.
15. AO Garba, SA Mousa. Bevasiranib for the treatment of wet, age-related macular degeneration, Ophthalmol. Eye Dis. 2010; 2: 75-83.
16. QD Nguyen, RA Schachar, CI Nduaka, M Sperling, AS Basile, KJ Klamerus, et al. Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema (the DEGAS study), Invest.Ophthalmol. Vis. Sci. 2012; 53: 7666-7674.
17. H Nakamura, SS Siddiqui, X Shen, AB Malik, JS Pulido, NM Kumar, et al. RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis, Mol. Vis. 2004; 10: 703-711.
18. H Ye, Y Qian, M Lin, Y Duan, X Sun, Y Zhuo, et al. Cationic nano-copolymers mediated IKKbeta targeting siRNA to modulate wound healing in a monkey model of glaucoma filtration surgery, Mol. Vis. 2010; 16: 2502-2510.
19. T Martinez, MV Gonzalez, I Roehl, N Wright, C Paneda, AI Jimenez. In vitro and in vivo efficacy of SYL040012, a novel siRNA compound for treatment of glaucoma, Mol. Ther. 2014; 22: 81-91.
20. A Thakur, S Fitzpatrick, A Zaman, K Kugathasan, B Muirhead, G Hortelano, et al. Strategies for ocular siRNA delivery: potential and limitations of nonviral nanocarriers, J. Biol. Eng. 2012; 6: 7.
21. A Rodriguez-Aguayo, A Chavez-Reyes, G Lopez-Berestein, AK Sood. RNAi in cancer therapy, in: K Cheng, RI Mahato (Eds.), Advanced Delivery and Therapeutic Applications of RNAi, John Wiley and Sons, Ltd .2013; 271-307.
22. ME Davis, JE Zuckerman, CHJ Choi, D Seligson, A Tolcher, CA Alabi, et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles, Nature. 2010; 464: 1067-1070.
23. NS Gandhi, RK Tekade, MB Chougule. Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances, J. Control. Release. 2014; 194: 238-256.
24. DR Senger, SJ Galli, AM Dvorak, CA Perruzzi, VS Harvey, HF Dvorak. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid, Science. 1983; 219: 983-985.
25. MD Cole. The myc oncogene: its role in transformation and differentiation, Annu. Rev. Genet.1986; 20: 361-384.
26. EP Sulman, XX Tang, C Allen, JA Biegel, DE Pleasure, GM Brodeur, et al. ECK a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers, Genomics. 1997; 40: 371-374.
27. UR Rapp, MD Goldsborough, GE Mark, TI Bonner, J Groffen, FH Reynolds Jr, et al. Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus, Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4218- 4222.
28. U Holtrich, G Wolf, A Brauninger, T Karn, B Bohme, H Rubsamen- Waigmann, et al. Induction and down-regulation of PLK, a human serine/ threonine kinase expressed in proliferating cells and tumors, Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1736-1740.
29. MG Lee, P Nurse. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2, Nature. 1987; 327: 31-35.
30. RL Juliano, V Ling. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants, Biochim. Biophys. Acta. 1976; 455: 152-162.
31. Y Chen, H Gu, DS Zhang, F Li, T Liu, W Xia. Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier, Biomaterials. 2014; 35: 10058-10069.
32. X Liu, W Wang, D Samarsky, L Liu, Q Xu, W Zhang, et al. Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA, Nucleic Acids Res. 2015; 42: 11805-11817.
33. Y Chen, SR Bathula, Q Yang, L Huang. Targeted nanoparticles deliver siRNA to melanoma, J. Investig.Dermatol. 2010; 130: 2790-2798.
34. Y Chen, X Zhu, X Zhang, B Liu, L Huang. Nanoparticles modified with tumortargeting scFv deliver siRNA and miRNA for cancer therapy, Mol. Ther. 2010; 18: 1650-1656.
35. CN Landen Jr., A Chavez-Reyes, C Bucana, R Schmandt, MT Deavers, G Lopez-Berestein, et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery, Cancer Res. 2005; 65: 6910-6918.
36. ST Chou, Q Leng, P Scaria, JD Kahn, LJ Tricoli, M Woodle, et al. Surfacemodified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition, Biomacromolecules. 2013; 14: 752-760.
37. Y Sakurai, H Hatakeyama, Y Sato, M Hyodo, H Akita, H Harashima. Gene silencing via RNAi and siRNA quantification in tumor tissue using MEND, a liposomal siRNA delivery system, Mol. Ther. 2013; 21: 1195-1203.
38. Y Liu, YH Zhu, CQ Mao, S Dou, S Shen, ZB Tan, et al. Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEGPLA nanoparticles, J. Control. Release. 2014; 192: 114-121.
39. L Zhu, F Perche, T Wang, VP Torchilin. Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs, Biomaterials. 2014; 35: 4213-4222.
40. JY Yhee, S Song, SJ Lee, SG Park, KS Kim, MG Kim, Set al. Cancer-targeted MDR-1 siRNA delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance, J. Control. Release. 2015; 198: 1-9.
41. G Neufeld, T Cohen, S Gengrinovitch, Z Poltorak. Vascular endothelial growth factor (VEGF) and its receptors, FASEB J. 1999; 13: 9-22.
42. D Zhuang, S Mannava, V Grachtchouk, WH Tang, S Patil, JA Wawrzyniak. et al. C-MYC overexpression is required for continuous suppression of oncogene-induced senescence in melanoma cells, Oncogene. 2008; 27: 6623-6634.
43. RW Johnstone, AA Ruefli, SW Lowe. Apoptosis: a link between cancer genetics and chemotherapy, Cell. 2002; 108: 153-164.
44. PH Thaker, M Deavers, J Celestino, A Thornton, MS Fletcher, CN Landen, et al. Sood, EphA2 expression is associated with aggressive features in ovarian carcinoma, Clin. Cancer Res. 2004; 10: 5145-5150.
45. M Nakamoto, AD Bergemann. Diverse roles for the Eph family of receptor tyrosine kinases in carcinogenesis, Microsc. Res. Tech. 2002; 59: 58-67.
46. F Eckerdt, J Yuan, K Strebhardt. Polo-like kinases and oncogenesis, Oncogene. 2005; 24: 267-276.
47. K Ando, T Ozaki, H Yamamoto, K Furuya, M Hosoda, S Hayashi, et al. Polo-like kinase 1 (Plk1) inhibits p53 function by physical interaction and phosphorylation, J. Biol. Chem. 2004; 279: 25549-25561.
48. M Malumbres, M Barbacid. Mammalian cyclin-dependent kinases, Trends Biochem. Sci. 2005; 30: 630-641.
49. M Malumbres, M Barbacid. To cycle or not to cycle: a critical decision in cancer, Nat. Rev. Cancer. 2001; 1: 222-231.
50. M. Malumbres. Cyclin-dependent kinases,Genome Biol. 2014;15: 122.
51. P Bonelli, FM Tuccillo, A Borrelli, A Schiattarella, FM Buonaguro. CDK/ CCN and CDKI alterations for cancer prognosis and therapeutic predictivity, Biomed. Res. Int. 2014; 2014: 361020.
52. Breakthrough Therapies.
53. G Ambrosini, C Adida, DC Altieri. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma, Nat. Med. 1997; 3: 917-921.
54. DC Altieri. Survivin, versatile modulation of cell division and apoptosis in cancer, Oncogene. 2003; 22: 8581-8589.
55. G Bradley, V Ling. P-glycoprotein, multidrug resistance and tumor progression, Cancer Metastasis Rev. 1994; 13: 223-233.
56. M Abbasi, A Lavasanifar, H Uludag. Recent attempts at RNAi-mediated Pglycoprotein downregulation for reversal of multidrug resistance in cancer, Med. Res. Rev. 2013; 33: 33-53.
57. C Lorenzer, M Dirin, AM Winkler, V Baumann, J Winkler. Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics, J. Control. Release. 2015; 203: 1-15.
58. P Arbuthnot, A Ely, MS Weinberg. Hepatic delivery of RNA interference activators for therapeutic application, Curr. Gene Ther. 2009; 9: 91-103.
59. A Erez, OA Shchelochkov, SE Plon, F Scaglia, B Lee. Insights into the pathogenesis and treatment of cancer from inborn errors of metabolism, Am. J. Hum. Genet. 2011; 88: 402-421.
60. MRS Alix Scholer-Dahirel, Alice Loo, Linda Bagdasarian, Ronald Meyer, Ribo Guo, Steve Woolfenden, et al. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/β-catenin signaling, Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17135-17140.
61. E Raskopf, A Vogt, T Sauerbruch, V Schmitz. siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo, J. Hepatol. 2008; 49: 977-984.
62. K Zhang, K Chu, X Wu, H Gao, J Wang, YC Yuan, et al. Amplification of FRS2 and activation of FGFR/FRS2 signaling pathway in high-grade liposarcoma, Cancer Res. 2013; 73: 1298-1307.
63. PG Rychahou, LN Jackson, SR Silva, S Rajaraman, BM Evers. Targetedmolecular therapy of the PI3K pathway: therapeutic significance of PI3K subunit targeting in colorectal carcinoma, Ann. Surg. 2006; 243: 833- 842.
64. H Clevers. Wnt/beta-catenin signaling in development and disease, Cell. 2006; 127: 469-480.
65. AO Kaseb, A Hanbali, M Cotant, MM Hassan, I Wollner, PA.Philip. Vascular endothelial growth factor in the management of hepatocellular carcinoma: a review of literature, Cancer. 2009; 115: 4895-4906.
66. VP Eswarakumar, I Lax, J Schlessinger. Cellular signaling by fibroblast growth factor receptors, Cytokine Growth Factor Rev. 2005; 16: 139-149.
67. DA Fruman C Rommel. PI3K and cancer: lessons, challenges and opportunities, Nat. Rev. Drug Discov. 2014; 13: 140-156.
68. P Huang, X Xu, L Wang, B Zhu, X Wang, J Xia. The role of EGF– EGFR signaling pathway in hepatocellular carcinoma inflammatory microenvironment, J. Cell. Mol. Med. 2014; 18: 218-230.
69. S Whittaker, R Marais, AX Zhu. The role of signaling pathways in the development and treatment of hepatocellular carcinoma, Oncogene. 2010; 29: 4989-5005.
70. G Szabo, S Bala. MicroRNAs in liver disease, Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 542-552.
71. F Azam, A Koulaouzidis. Hepatitis B virus and hepatocarcinogenesis, Ann. Hepatol. 2008; 7: 125-129.
72. AB Cefalu, JP Pirruccello, D Noto, S Gabriel, V Valenti, N Gupta, et al. A novel APOB mutation identified by exome sequencing cosegregates with steatosis, liver cancer, and hypocholesterolemia, Arterioscler.Thromb.Vasc. Biol. 2013; 33: 2021-2025.
73. XQ Wang, W Zhang, EL Lui, Y Zhu, P Lu, X Yu, et al. Notch-Snail-E-cadherin pathway in metastatic hepatocellular carcinoma, Int. J. Cancer. 2012; 131: E163-E172.
74. M Riaz, M Idrees, H Kanwal, F Kabir. An overview of triple infection with hepatitis B, C and D viruses, Virol. J. 2011; 8: 368.
75. Hepatitis B information for public.
76. D Halegoua-De Marzio, HW Hann. Then and now: the progress in hepatitis B treatment over the past 20 years, World J. Gastroenterol. 2014; 20: 401-413.
77. CI Wooddell, DB Rozema, M Hossbach, M John, HL Hamilton, Q Chu, et al. Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection, Mol. Ther. 2013; 21: 973-985.
78. S Carmona, MR Jorgensen, S Kolli, C Crowther, FH Salazar, PL Marion, et al. Controlling HBV replication in vivo by intravenous administration of triggered PEGylated siRNA-nanoparticles, Mol. Pharm. 2009; 6: 706-717.
79. SI Kim, D Shin, TH Choi, JC Lee, GJ Cheon, KY Kim, et al. Systemic and specific delivery of small interfering RNAs to the livermediated by apolipoprotein A-I, Mol. Ther. 2007; 15: 1145-1152.
80. M Konishi, CH Wu, GY Wu. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line, Hepatology. 2003; 38: 842-850.
81. G Li, G Jiang, J Lu, S Chen, L Cui, J Jiao, et al. Inhibition of hepatitis B virus cccDNA by siRNA in transgenic mice, Cell Biochem. Biophys. 2014; 69: 649-654.
82. GQ Li, HX Gu, D Li, WZ Xu. Inhibition of hepatitis B virus cccDNA replication by siRNA, Biochem. Biophys. Res. Commun. 2007; 355: 404-408.
83. D Grimm. All for one, one for all: new combinatorial RNAi therapies combat hepatitis C virus evolution, Mol. Ther. 2012; 20: 1661-1663.
84. TJ Liang, MG Ghany. Current and future therapies for hepatitis C virus infection, N. Engl. J. Med. 2013; 368: 1907-1917.
85. M Korf, D Jarczak, C Beger, MP Manns, M Kruger. Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors, J. Hepatol. 2005; 43: 225-234.
86. T Watanabe, T Umehara, F Yasui, SI Nakagawa, J Yano, T Ohgi. et al. Liver target delivery of small interfering RNA to the HCV gene by lactosylated cationic liposome, J. Hepatol. 2007; 47: 744-750.
87. H Ma, A Dallas, H Ilves, J Shorenstein, I MacLachlan, K Klumpp, et al. Formulated minimal-length synthetic small hairpin RNAs are potent inhibitors of hepatitis C virus in mice with humanized livers, Gastroenterology. 2014; 146: 63-66.e65.
88. PK Chandra, AK Kundu, S Hazari, S Chandra, L Bao, T Ooms, et al. Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes, Mol. Ther. 2012; 20: 1724-1736.
89. SI Kim, D Shin, H Lee, BY Ahn, Y Yoon, M Kim. Targeted delivery of siRNA against hepatitis C virus by apolipoprotein A–I-bound cationic liposomes, J. Hepatol. 2009; 50: 479-488.
90. M Ansar, UA Ashfaq, I Shahid, MT Sarwar, T Javed, S Rehman, et al. Inhibition of full length hepatitis C virus particles of 1a genotype through small interference RNA, Virol. J. 2011; 8: 203.
91. TW Geisbert, ACH Lee, M Robbins, JB Geisbert, AN Honko, V Sood, et al. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study, Lancet. 2010; 375: 1896-1905.
92. TW Geisbert, LE Hensley, E Kagan, EZ Yu, JB Geisbert, K Daddario- DiCaprio, et al. Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference, J. Infect. Dis. 2006; 193: 1650-1657.
93. A Shlomai, Y Shaul. Inhibition of hepatitis B virus expression and replication by RNA interference, Hepatology. 2003; 37: 764-770.
94. OM Merkel, I Rubinstein, T Kissel. siRNA delivery to the lung: what’s new? Adv. Drug Deliv. Rev. 2014; 75: 112-128.
95. C Kelly, AB Yadav, PJ McKiernan, CM Greene, SA Cryan. RNAi in respiratory diseases, in: K. Cheng, R.I. Mahato (Eds.), Advanced Delivery and Therapeutic Applications of RNAi, John Wiley and Sons, Ltd. 2013; 391- 416.
96. M Grzelinski, O Pinkenburg, T Buch, M Gold, S Stohr, H Kalwa, et al. Critical role of G(alpha)12 and G(alpha)13 for human small cell lung cancer cell proliferation in vitro and tumor growth in vivo, Clin. Cancer Res. 2010; 16: 1402-1415.
97. DE Zamora-Avila, P Zapata-Benavides, MA Franco-Molina, S Saavedra- Alonso, LM Trejo-Avila, D Resendez-Perez, et al. WT1 gene silencing by aerosol delivery of PEI–RNAi complexes inhibits B16–F10 lung metastases growth, Cancer Gene Ther. 2009; 16: 892-899.
98. Q Ge, L Filip, A Bai, T Nguyen, HN Eisen, J Chen. Inhibition of influenza virus production in virus-infected mice by RNA interference, Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8676-8681.
99. M Thomas, JJ Lu, Q Ge, C Zhang, J Chen, AM Klibanov. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung, Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5679- 5684.
100. V Bitko, A Musiyenko, O Shulyayeva, S Barik. Inhibition of respiratory viruses by nasally administered siRNA, Nat. Med. 2005; 11: 50-55.
101. KL Clark, SA Hughes, P Bulsara, J Coates, K Moores, J Parry, et al. Pharmacological characterization of a novel ENaCalpha siRNA (GSK2225745) with potential for the treatment of cystic fibrosis, Mol. Ther.- Nucleic Acids. 2013; 2: e65.
102. JH Seong, KM Lee, ST Kim, SE Jin, CK Kim. Polyethylenimine-based antisense oligodeoxynucleotides of IL-4 suppress the production of IL-4 in a murine model of airway inflammation, J. Gene. Med. 2006; 8: 314-323.
103. TN Lively, K Kossen, A Balhorn, T Koya, S Zinnen, K Takeda, et al. Effect of chemically modified IL-13 short interfering RNA on development of airway hyperresponsiveness in mice, J. Allergy Clin. Immunol. 2008; 121: 88-94.
104. M Zheng, D Librizzi, A Kilic, Y Liu, H Renz, OM Merkel, et al. Enhancing in vivo circulation and siRNA delivery with biodegradable polyethyleniminegraftpolycaprolactone- block-poly(ethylene glycol) copolymers, Biomaterials. 2012; 33: 6551-6558.
105. Y Oe, RJ Christie, M Naito, SA Low, S Fukushima, K Toh, et al. Activelytargeted polyion complex micelles stabilized by cholesterol and disulfide cross-linking for systemic delivery of siRNA to solid tumors, Biomaterials. 2014; 35: 7887-7895.
106. Y Matsumoto, T Nomoto, H Cabral, Y Matsumoto, S Watanabe, RJ Christie, et al. Direct and instantaneous observation of intravenously injected substances using intravital confocal micro-videography, Biomed. Opt. Express. 2010; 1: 1209-1216.
107. FM van de Water, OC Boerman, AC Wouterse, JG Peters, FG Russel, R Masereeuw. Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules, Drug Metab. Dispos. 2006; 34: 1393-1397.
108. J Christensen, K Litherland, T Faller, E van de Kerkhof, F Natt, J Hunziker, et al. Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice, Drug Metab.Dispos. 2013; 41: 1211-1219.
109. S Gao, F Dagnaes-Hansen, EJ Nielsen, J Wengel, F Besenbacher, KA Howard, et al. The effect of chemicalmodification and nanoparticle formulation on stability and biodistribution of siRNA in mice, Mol. Ther. 2009; 17: 1225-1233.
110. J Christensen, K Litherland, T Faller, E van de Kerkhof, F Natt, J Hunziker, et al. Biodistribution and metabolism studies of lipid nanoparticleformulated internally [3H]-labeled siRNA in mice, Drug Metab. Dispos. 2014; 42: 431- 440.
111. G Ozcan, B Ozpolat, RL Coleman, AK Sood, G Lopez-Berestein. Preclinical and clinical development of siRNA-based therapeutics, Adv. Drug Deliv. Rev. 2015; 87: 108-119.
112. J Tabernero, GI Shapiro, PM LoRusso, A Cervantes, GK Schwartz, GJ Weiss, et al. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement, Cancer Discov. 2013; 3: 406.417.
113. B Schultheis, D Strumberg, A Santel, C Vank, F Gebhardt, O Keil, et al. First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors, J. Clin. Oncol. 2014; 32: 4141-4148.
114. DW Bartlett, H Su, IJ Hildebrandt, WA Weber, ME Davis. Impact of tumorspecific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging, Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15549-15554.
115. JE Zuckerman, I Gritli, A Tolcher, JD Heidel, D Lim, R Morgan, et al. Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA, Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 11449-11454.
116. RZ Yu, B Baker, A Chappell, RS Geary, E Cheung, AA Levin. Development of an ultrasensitive noncompetitive hybridization-ligation enzymelinked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma, Anal.Biochem. 2002; 304: 19-25.
117. JE Zuckerman, CH Choi, H Han, ME Davis. Polycation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane, Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 3137-3142.
118. Y. Chen, J.J. Wu, L. Huang, Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy, Mol. Ther. 2010; 18: 828-834.
119. MS Lee, JE Lee, E Byun, NW Kim, K Lee, H Lee, et al. Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopahyaluronic acid conjugate, J. Control. Release. 2014; 192: 122-130.
120. F Pittella, H Cabral, Y Maeda, P Mi, S Watanabe, H Takemoto, et al. Systemic siRNA delivery to a spontaneous pancreatic tumor model in transgenic mice by PEGylated calcium phosphate hybrid micelles, J. Control. Release. 2014; 178: 18-24.
121. S Son, S Song, SJ Lee, S Min, SA Kim, JY Yhee, et al. Self-crosslinked human serum albumin nanocarriers for systemic delivery of polymerized siRNA to tumors, Biomaterials. 2013; 34: 9475-9485.
122. B Shi, E Keough, A Matter, K Leander, S Young, E Carlini, et al. Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery, J. Histochem.Cytochem. 2011; 59: 727- 740.
123. KY Choi, OF Silvestre, X Huang, KH Min, GP Howard, N Hida, et al. Versatile RNA interference nanoplatform for systemic delivery of RNAs, ACS Nano. 2014; 8: 4559-4570.
124. SA Jensen, ES Day, CH Ko, LA Hurley, JP Luciano, FM Kouri, et al. Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma, Sci. Transl. Med. 2013; 5: 209-152.
125. A Schlegel, C Largeau, P Bigey, M Bessodes, K Lebozec, D Scherman, et al. Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes, J. Control. Release. 2011; 152: 393-401.
126. J Gao, H Chen, Y Yu, J Song, H Song, X Su, et al. Inhibition of hepatocellular carcinoma growth using immunoliposomes for co-delivery of adriamycin and ribonucleotide reductaseM2 siRNA, Biomaterials. 2013; 34: 10084-10098.
127. XB Xiong, A Lavasanifar. Traceable multifunctional micellar nanocarriers for cancer-targeted co-delivery of MDR-1 siRNA and doxorubicin, ACS Nano. 2011; 5: 5202-5213.
128. G Sass, P Leukel, V Schmitz, E Raskopf, M Ocker, D Neureiter, et al. Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice, Int. J. Cancer. 2008; 123: 1269-1277.
129. Jinho Park, Joonyoung Park, Yihua Pei, Jun Xu, Yoon Yeo. Pharmacokinetics and biodistribution of recently-developed siRNA nanomedicines. Advanced Drug Delivery Reviews. 2016; 104: 93-109.
130. M Kornek, V Lukacs-Kornek, A Limmer, E Raskopf, U Becker, M Klockner, et al. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)- formulated, immune-stimulatory vascular endothelial growth factor a small interfering RNA (siRNA) increases antitumoral efficacy inmurine orthotopic hepatocellular carcinoma with liver fibrosis, Mol. Med. 2008; 14: 365-373.
131. L Li, R Wang, D Wilcox, A Sarthy, X Lin, X Huang, et al. Developing lipid nanoparticle-based siRNA therapeutics for hepatocellular carcinoma using an integrated approach, Mol. Cancer Ther. 2013; 12: 2308-2318.
132. J Gao, Y Yu, Y Zhang, J Song, H Chen, W Li, et al. EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma, Biomaterials. 2012; 33: 270-282.
133. DV Morrissey, JA Lockridge, L Shaw, K Blanchard, K Jensen, W Breen, et al. Potent and persistent in vivo anti-HBV activity of chemicallymodified siRNAs, Nat. Biotechnol. 2005; 23: 1002-1007.
134. DV Morrissey, K Blanchard, L Shaw, K Jensen, JA Lockridge, B Dickinson, et al. Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication, Hepatology. 2005; 41: 1349-1356.
135. M Mével, N Kamaly, S Carmona, MH Oliver, MR Jorgensen, C Crowther, et al. DODAG; a versatile new cationic lipid that mediates efficient delivery of pDNA and siRNA, J. Control. Release. 2010; 143: 222-232.
136. H Lee, SI Kim, D Shin, Y Yoon, TH Choi, GJ Cheon, et al. Hepatic siRNA delivery using recombinant human apolipoprotein A–I in mice, Biochem. Biophys. Res. Commun. 2009; 378: 192-196.
137. Study of ARC-520 in Patients with Chronic Hepatitis B Virus.
138. Safety, Tolerability and Pharmacokinetic First in Human (FIH) Study for Intravenous (IV) TKM-100802.
139. CS Kraft, AL Hewlett, S Koepsell, AM Winkler, CJ Kratochvil, L Larson, et al. The use of TKM-100802 and convalescent plasma in 2 patients with Ebola virus disease in the United States, Clin.Infect. Dis. 2015; 61: 496-502.
140. M Gunther, J Lipka, A Malek, D Gutsch, W Kreyling, A Aigner. Polyethylenimines for RNAi-mediated gene targeting in vivo and siRNA delivery to the lung, Eur. J. Pharm. Biopharm. 2011; 77: 438-449.
141. ME Bonnet, JB Gossart, E Benoit, M Messmer, O Zounib, V Moreau, et al. Systemic delivery of sticky siRNAs targeting the cell cycle for lung tumor metastasis inhibition, J. Control. Release. 2013; 170: 183-190.
142. AL Bolcato-Bellemin, ME Bonnet, G Creusat, P Erbacher, JP Behr. Sticky overhangs enhance siRNA-mediated gene silencing, Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16050-16055.
143. J McCaskill, R Singhania, M Burgess, R Allavena, S Wu, A Blumenthal, et al. Efficient biodistribution and gene silencing in the lung epithelium via intravenous liposomal delivery of siRNA, Mol. Ther. Nucleic Acids. 2013; 2: e96.
144. V Fehring, U Schaeper, K Ahrens, A Santel, O Keil, M Eisermann, et al. Delivery of therapeutic siRNA to the lung endothelium via novel lipoplex formulation DACC, Mol. Ther. 2014; 22: 811-820.
145. KJ Polach, M Matar, J Rice, G Slobodkin, J Sparks, R Congo, et al. Delivery of siRNA to the mouse lung via a functionalized lipopolyamine, Mol. Ther. 2012; 20: 91-100.
146. JE Dahlman, C Barnes, OF Khan, A Thiriot, S Jhunjunwala, TE Shaw, et al. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight, Nat. Nanotechnol. 2014; 9: 648-655.
147. OB Garbuzenko, M Saad, S Betigeri, M Zhang, AA Vetcher, VA Soldatenkov, et al. Intratracheal versus intravenous liposomal delivery of siRNA, antisense oligonucleotides and anticancer drug, Pharm. Res. 2009; 26: 382-394.
148. OM Merkel, A Beyerle, D Librizzi, A Pfestroff, TM Behr, B Sproat, et al. Nonviral siRNA delivery to the lung: investigation of PEG-PEI polyplexes and their in vivo performance, Mol. Pharm. 2009; 6: 1246-1260.
149. B Gutbier, SM Kube, K Reppe, A Santel, C Lange, J Kaufmann, et al. RNAimediated suppression of constitutive pulmonary gene expression by small interfering RNA in mice, Pulm. Pharmacol.Ther. 2010; 23: 334-344.
150. A Wittrup, J Lieberman. Knocking down disease: a progress report on siRNA therapeutics, Nat. Rev. Genet. 2015; 16: 543-552.
151. AL Jackson, J Burchard, D Leake, A Reynolds, J Schelter, J Guo, et al. Positionspecific chemicalmodification of siRNAs reduces “off-target” transcript silencing, RNA. 2006; 12: 1197-1205.
152. L Huang, J Jin, P Deighan, E Kiner, L McReynolds, J Lieberman. Efficient and specific gene knockdown by small interfering RNAs produced in bacteria, Nat. Biotechnol. 2013; 31: 350-356.
153. S Loisel, C Le Gall, L Doucet, C Ferec, V Floch. Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes, Hum. Gene Ther. 2001; 12: 685-696.
154. JD Tousignant, AL Gates, LA Ingram, CL Johnson, JB Nietupski, SH Cheng, et al. Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice, Hum. Gene Ther. 2000; 11: 2493-2513.
155. W Yan, W Chen, L Huang. Mechanism of adjuvant activity of cationic liposome: phosphorylation of a MAP kinase, ERK and induction of chemokines, Mol. Immunol. 2007; 44: 3672-3681.
156. DP Vangasseri, Z Cui, W Chen, DA Hokey, LD Falo Jr., L Huang. Immunostimulation of dendritic cells by cationic liposomes, Mol. Membr. Biol. 2006; 23: 385-395.
157. T Tanaka, A Legat, E Adam, J Steuve, JS Gatot, M Vandenbranden, L. et al. DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4, Eur. J. Immunol. 2008; 38: 1351-1357.
158. D Fischer, T Bieber, Y Li, HP Elsässer, T Kissel. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity, Pharm. Res. 1999; 16: 1273-1279.
159. MA Islam, TE Park, B Singh, S Maharjan, J Firdous, MH Cho, et al. Major degradable polycations as carriers for DNA and siRNA, J. Control. Release. 2014; 193: 74-89.
160. Ming-Nan Chien, Po-Sheng Yang, Jie-Jen Lee, Tao-Yeuan Wang, Yi- Chiung Hsu, Shih-Ping Cheng. Recurrence-associated genes in papillary thyroid cancer: An analysis of data from The Cancer Genome Atlas. Surgery. 2017; 161: 1642-1650.
161. Mao Y, Xing M. Recent incidences and differential trends of thyroid cancer in the USA. Endocr Relat Cancer. 2016; 23: 313-322.
162. Grogan RH, Kaplan SP, Cao H, Weiss RE, Degroot LJ, Simon CA, et al. A study of recurrence and death from papillary thyroid cancer with 27 years of median follow-up. Surgery. 2013; 154:1436-1446.
163. American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009; 19: 1167-1214.
164. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, et al. 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016; 26:1-133.
165. Cheng SP, Hsu YC, Liu CL, Liu TP, Chien MN, Wang TY, et al. Significance of allelic percentage of BRAF c.1799T > A (V600E) mutation in papillary thyroid carcinoma. Ann Surg Oncol. 2014; 21: S619-626.
166. Gandolfi G, Sancisi V, Piana S, Ciarrocchi A. Time to reconsider the meaning of BRAF V600E mutation in papillary thyroid carcinoma. Int J Cancer. 2015; 137:1001-1011.
167. Liu R, Xing M. TERT promoter mutations in thyroid cancer. Endocr Relat Cancer. 2016; 23: R143-155.
168. Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014; 159: 676-690.
169. Wang J, Duncan D, Shi Z, Zhang B. WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013. Nucleic Acids Res. 2013; 41: W77-83.
170. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4: 44-57.
171. Chang YC, Hsu YC, Liu CL, Huang SY, Hu MC, Cheng SP. Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway. PLoS One. 2014; 9: e89563.
172. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016; 44: D457-462.
173. Lee F, Lee JJ, Jan WC, Wu CJ, Chen HH, Cheng SP. Molecular pathways associated with transcriptional alterations in hyperparathyroidism. Oncol Lett. 2016; 12: 621-626.
174. Chai YJ, Yi JW, Jee HG, Kim YA, Kim JH, Xing M, et al. Significance of the BRAF mRNA expression level in papillary thyroid carcinoma: an analysis of The Cancer Genome Atlas data. PLoS One. 2016; 11: e0159235.
175. Rivera M, Ghossein RA, Schoder H, Gomez D, Larson SM, Tuttle RM. Histopathologic characterization of radioactive iodine-refractory fluorodeoxyglucose-positron emission tomography-positive thyroid carcinoma. Cancer 2008; 113: 48-56.
176. Viola D, Valerio L, Molinaro E, Agate L, Bottici V, Biagini A, et al. Treatment of advanced thyroid cancer with targeted therapies: ten years of experience. Endocr Relat Cancer. 2016; 23: R185-205.
177. Mian C, Barollo S, Pennelli G, Pavan N, Rugge M, Pelizzo MR, et al. Molecular characteristics in papillary thyroid cancers (PTCs) with no 131I uptake. Clin Endocrinol (Oxf) 2008; 68: 108-116.
178. Sabra MM, Dominguez JM, Grewal RK, Larson SM, Ghossein RA, Tuttle RM, et al. Clinical outcomes and molecular profile of differentiated thyroid cancers with radioiodine-avid distant metastases. J Clin Endocrinol Metab. 2013; 98: E829-836.
179. Portulano C, Paroder-Belenitsky M, Carrasco N. The Na+/Isymporter (NIS): mechanism and medical impact. Endocr Rev. 2014; 35: 106-149.
180. Spitzweg C, Bible KC, Hofbauer LC, Morris JC. Advanced radioiodinerefractory differentiated thyroid cancer: the sodium iodide symporter and other emerging therapeutic targets. Lancet Diabetes Endocrinol 2014; 2: 830-842.
181. Handkiewicz-Junak D, Roskosz J, Hasse-Lazar K, Szpak- Ulczok S, Puch Z, Kukulska A, et al. 13-cis-retinoic acid redifferentiation therapy and recombinant human thyrotropin-aided radioiodine treatment of non- Functional metastatic thyroid cancer: a single-center, 53-patient phase 2 study. Thyroid Res 2009; 2: 8.
182. Czajka AA, Wojcicka A, Kubiak A, Kotlarek M, Bakula-Zalewska E, Koperski L, et al. Family of microRNA-146 regulates RARb in papillary thyroid carcinoma. PLoS One. 2016; 11: e0151968.
183. Shen CT, Qiu ZL, Song HJ, Wei WJ, Luo QY. miRNA-106a directly targeting RARB associates with the expression of Na(+)/I(-) symporter in thyroid cancer by regulating MAPK signaling pathway. J Exp Clin Cancer Res 2016; 35: 101.
184. Ho AL, Grewal RK, Leboeuf R, Sherman EJ, Pfister DG, Deandreis D, et al. Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer. N Engl J Med. 2013; 368: 623-32.
185. Rothenberg SM, McFadden DG, Palmer EL, Daniels GH, Wirth LJ. Redifferentiation of iodine-refractory BRAF V600E-mutant metastatic papillary thyroid cancer with dabrafenib. Clin Cancer Res. 2015; 21: 1028- 1035.
186. Brose MS, Cabanillas ME, Cohen EE, Wirth LJ, Riehl T, Yue H, et al. Vemurafenib in patients with BRAF(V600E)-positive metastatic or unresectable papillary thyroid cancer refractory to radioactive iodine: a nonrandomised, multicentre, open-label, phase 2 trial. Lancet Oncol. 2016; 17: 1272-1282.
187. Ranjbari N, Rahim F. The Ki-67/MIB-1 index level and recurrence of papillary thyroid carcinoma. Med Hypotheses 2013; 80: 311-314.
188. Albero A, Lopez JE, Torres A, de la Cruz L, Martin T. Effectiveness of chemotherapy in advanced differentiated thyroid cancer: a systematic review. Endocr Relat Cancer. 2016; 23: R71-84.
189. Peng L, Bian XW, Li DK, Xu C, Wang GM, Xia QY, et al. Large-scale RNAseq transcriptome analysis of 4043 cancers and 548 normal tissue controls across 12 TCGA cancer types. Sci Rep. 2015; 5: 13413.
190. Gatzidou E, Michailidi C, Tseleni-Balafouta S, Theocharis S. An epitome of DNA repair related genes and mechanisms in thyroid carcinoma. Cancer Lett. 2010; 290: 139-147.
191. Estrada-Bernal A, Chatterjee M, Haque SJ, Yang L, Morgan MA, Kotian S, et al. MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways. Cell Cycle. 2015; 14: 3713-3724.
192. Bovelstad HM, Nygard S, Storvold HL, Aldrin M, Borgan O, Frigessi A, et al. Predicting survival from microarray data–a comparative study. Bioinformatics 2007; 23: 2080-2087.
193. Brennan K, Holsinger C, Dosiou C, Sunwoo JB, Akatsu H, Haile R, et al. Development of prognostic signatures for intermediate-risk papillary thyroid cancer. BMC Cancer. 2016; 16: 736.
194. Loutfy H. Madkour Book: Reactive Oxygen Species (ROS), Nanoparticles, and Endoplasmic Reticulum (ER) Stress-Induced Cell Death Mechanisms. Paperback ISBN: 9780128224816 Imprint: Academic Press Published. 2020.
195. Loutfy H. Madkour Book: Nanoparticles Induce Oxidative and Endoplasmic Reticulum Antioxidant Therapeutic Defenses.
196. Loutfy H. Madkour Book: Nucleic Acids as Gene Anticancer Drug Delivery Therapy. 1st Edition .Publishing house: Elsevier. 2020.
197. Loutfy H. Madkour Book: Nanoelectronic Materials: Fundamentals and Applications (Advanced Structured Materials) 1st ed. 2019.

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Citation: Madkour LH. Pharmacokinetics, Bio Distribution and Therapeutic Applications of Recently-Developed siRNA and DNA Repair Genes Recurrence. Austin J Anal Pharm Chem. 2021; 8(2): 1135.

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