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Background: Estrogen receptors express in nearly 70% of breast cancers (ER-positive). Estrogen receptor alpha plays a fundamental role as a significant factor in breast cancer progression for the early selection of therapeutic approaches. Accordingly, there has been a surge of attention to non-invasive techniques, including circulating Cell-free DNA (ccfDNA) or Cell-Free DNA (cfDNA), to detect and track ESR1 genotype. Therefore, this study aimed to examine the diagnosis accuracy of ESR1 mutation detection by cell-free DNA in breast cancer patientsthrough a systematic review and comprehensive meta-analysis., Methods: PubMed, Embase, and Web of Science databases were searched up to 6 April 2022. Diagnostic studies on ESR1 measurement by cfDNA, which was confirmed using the tumour tissue biopsy, have been included in the study. The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were considered to analyse the data., Results: Out of 649 papers, 13 papers with 15 cohorts, including 389 participants, entered the meta-analyses. The comprehensive meta-analysis indicated a high sensitivity (75.52, 95% CI 60.19-90.85), specificity (88.20, 95% CI 80.99-95.40), and high accuracy of 88.96 (95% CI 83.23-94.69) for plasma ESR1. We also found a moderate PPV of 56.94 (95% CI 41.70-72.18) but a high NPV of 88.53 (95% CI 82.61-94.44). We also found an NLR of 0.443 (95% CI 0.09-0.79) and PLR of 1.60 (95% CI 1.20-1.99)., Conclusion: This systematic review and comprehensive meta-analysis reveal that plasma cfDNA testing exhibits high sensitivity and specificity in detecting ESR1 mutations in breast cancer patients. This suggests that the test could be a valuable diagnostic tool. It may serve as a dependable and non-invasive technique for identifying ESR1 mutations in breast cancer patients. However, more extensive research is needed to confirm its prognostic value., (© 2024. The Author(s).)

Next-generation sequencing (NGS) has been increasingly popular in genomics studies over the last decade, as new sequencing technology has been created and improved. Recently, NGS started to be used in clinical oncology to improve cancer therapy through diverse modalities ranging from finding novel and rare cancer mutations, discovering cancer mutation carriers to reaching specific therapeutic approaches known as personalized medicine (PM). PM has the potential to minimize medical expenses by shifting the current traditional medical approach of treating cancer and other diseases to an individualized preventive and predictive approach. Currently, NGS can speed up in the early diagnosis of diseases and discover pharmacogenetic markers that help in personalizing therapies. Despite the tremendous growth in our understanding of genetics, NGS holds the added advantage of providing more comprehensive picture of cancer landscape and uncovering cancer development pathways. In this review, we provided a complete overview of potential NGS applications in scientific and clinical oncology, with a particular emphasis on pharmacogenomics in the direction of precision medicine treatment options., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Long non-coding RNAs (lncRNAs) have been recently reported to be involved in the pathoetiology of Parkinson's disease (PD). Circulatory levels of lncRNAs might be used as markers for PD. In the present work, we measured expression levels of HULC , PVT1 , MEG3 , SPRY4-IT1 , LINC-ROR and DSCAM-AS1 lncRNAs in the circulation of patients with PD versus healthy controls. Expression of HULC was lower in total patients compared with total controls (Expression ratio (ER)=0.19, adjusted P value<0.0001) as well as in female patients compared with female controls (ER=0.071, adjusted P value=0.0004). Expression of PVT1 was lower in total patients compared with total controls (ER=0.55, adjusted P value=0.0124). Expression of DSCAM-AS1 was higher in total patients compared with total controls (ER=5.67, P value=0.0029) and in male patients compared with male controls (ER=9.526, adjusted P value=0.0024). Expression of SPRY4-IT was higher in total patients compared with total controls (ER=2.64, adjusted P value<0.02) and in male patients compared with male controls (ER=3.43, P value<0.03). Expression of LINC-ROR was higher in total patients compared with total controls (ER=10.36, adjusted P value<0.0001) and in both male and female patients compared with sex-matched controls (ER=4.57, adjusted P value=0.03 and ER=23.47, adjusted P value=0.0019, respectively). Finally, expression of MEG3 was higher in total patients compared with total controls (ER=13.94, adjusted P value<0.0001) and in both male and female patients compared with sex-matched controls (ER=8.60, adjusted P value<0.004 and ER=22.58, adjusted P value<0.0085, respectively). ROC curve analysis revealed that MEG3 and LINC-ROR have diagnostic power of 0.77 and 0.73, respectively. Other lncRNAs had AUC values less than 0.7. Expression of none of lncRNAs was correlated with age of patients, disease duration, disease stage, MMSE or UPDRS. The current study provides further evidence for dysregulation of lncRNAs in the circulation of PD patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Honarmand Tamizkar, Gorji, Gholipour, Hussen, Mazdeh, Eslami, Taheri and Ghafouri-Fard.)

Pancreatic cancer is one of the most deadly neoplasms and the seventh major cause of cancer-related deaths among both males and females. This cancer has a poor prognosis due to the lack of appropriate methods for early detection of cancer. Long non-coding RNAs (lncRNAs) have been recently found to influence the progression and initiation of pancreatic cancer. MACC1-AS1, LINC00976, LINC00462, LINC01559, HOXA-AS2, LINC00152, TP73-AS1, XIST, SNHG12, LUCAT1, and UCA1 are among the oncogenic lncRNAs in pancreatic cancer. On the other hand, LINC01111, LINC01963, DGCR5, MEG3, GAS5, and LINC00261 are among tumor suppressor lncRNAs in this tissue. In the current review, we summarize the roles of these two classes of lncRNAs in pancreatic cancer and discuss their potential as attractive diagnostic and prognostic biomarkers for pancreatic cancer. We also identified that the low expression of MEG3, LINC01963, and LINC00261 and the high expression of MACC1-AS1, LINC00462, LINC01559, and UCA1 were significantly correlated with worse survival in pancreatic cancer patients. Further research on these lncRNAs will provide new clues that could potentially improve the early diagnosis, prognostic prediction, and personalized treatments of patients with pancreatic cancer.

Breast cancer is the most common form of cancer in women around the world. The molecular mechanisms of this heterogeneous disease have been extensively investigated; but yet; It requires a lot of sensitive and specific markers for prognosis and early detection approaches. Non-protein coding RNAs known as lncRNAs have been reported in tumorigenic involvement so they can be used for therapeutic purposes. In the present study, the expression levels of CCAT1, PDCD4, PDCD4-AS1, and MEG3 LncRNA in adjacent tumor and breast tissue in 88 Iranian patients were evaluated by quantitative real-time PCR. CCAT1 was significantly expressed and PDCD4-AS1 decreased in tumor samples, PDCD4 and PDCD4-AS1 showed a positive correlation with each other, higher levels of PDCD4-AS1 were associated with better survival, tumor samples showed lower levels of PDCD4 in Showed comparisons with normal tissue. Our findings suggest that lncRNAs play an important role in controlling gene expression after transcription of major tumor suppressors or carcinogenic genes, leading to the development of triple-negative breast cancer (TNBC). In conclusion, this study investigated the prognostic role of lncRNA in breast cancer patients., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Gastric cancer (GC) is one of the most commonly diagnosed neoplasms in the world. A number of environmental and lifestyle factors, particularly chronic infection with Helicobacter pylori, have been found to partake in the pathogenesis of GC. The advent of high-throughput genome and transcriptome analysis has enhanced the knowledge about molecular mechanisms of the pathogenesis of GC. However, data regarding the expression of several circRNAs, such as circLMTK2, are not consistent. We explain the role of circRNAs in the development of GC. We searched databases for the newest publications using the terms gastric cancer and circRNA. Each circRNA alteration, downstream targets, its impacts on cancer cells, and the prognostic and diagnostic roles of these circRNAs have been discussed. Taken together, circRNAs can be putative biomarkers in GC and potential targets for the treatment of this cancer. Yet, this field is still in its infancy and needs further experiments for reaching the clinical application. As these transcripts are stable in circulation, they can be used in non-invasive methods of cancer detection and patients' follow-up., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Pancreatic cancer is a fatal cancer which is expected to exceed breast cancer as the third foremost source of cancer mortality by 2025. This cancer has been associated with several somatic genetic aberrations including mutations in the KRAS, CDKN2A/p16, TP53, and SMAD4. In addition, epigenetic alterations have been shown to affect development of this cancer. miRNAs are among the mostly appreciated epigenetic factors in this regard. Several oncomiRs such as miR-212, miR 506, miR-196b, miR-221-3p, miR-301a-3p, miR-23a and miR-29a have been found to promote proliferation of pancreatic cancer cells and block apoptotic pathways in these cells. On the other hand, miR-451a, miR-506, miR-142, miR-216b, miR-519d-3p, miR-1181, miR-340, miR-143-3p, miR-203a-3p, miR-455, miR-15a, miR-135a and miR-202 are among tumor suppressor miRNAs that modulate proliferation and cell cycle transition in these cells. In the current paper, we will discuss the role of oncomiRs and tumor suppressor miRNAs in the evolution of pancreatic cancer. Moreover, we will summarize the application of miRNAs as diagnostic and prognostic markers in pancreatic cancer. These studies have shown the ability of miRNAs to be served as non-invasive markers for pancreatic cancer., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Lung cancer is among the leading causes of cancer mortality and incidence in both sexes. Different classes of transcripts have been proposed as molecular markers in this type of cancer. Circular RNAs (circRNAs) are a group of transcripts with circular enclosed and stable configuration. These transcripts are stable in the blood, thus can be used as markers for detection of disorders. Moreover, dysregulation of circRNAs in the affected tissues of patients with different cancers shows their possible roles in the carcinogenesis. Several circRNAs including circPRKC1, circFGFR1, hsa-circ-0020123 and circTP63 have been found to be up-regulated in lung cancer samples. Meanwhile, cir-ITCH, hsa&#95;circ&#95;100395, hsa&#95;circ&#95;0033155, circRNF13, circNOL10, circ-UBR5, circPTK2 and circCRIM1 have been shown to be down-regulated in lung cancer tissues compared with noncancerous counterparts. Finally, prognostic values of circPRKC1, circFGFR1, has-circ-00120123, circTP63, circ&#95;0067934, CDR1as, hsa&#95;circRN&#95;103809 and some other circRNAs have been appraised in lung cancer. In the current manuscript, we describe the impact and utility of circRNAs in the pathology of lung cancer., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Circular RNAs (circRNAs) are a group of regulatory non-coding transcripts, which partake in the pathobiology of hepatocellular carcinoma (HCC). Numerous micro-array based investigations have discovered aberrant expression of circRNAs in HCC samples in comparison with para-cancerous sections. Furthermore, a number of in vitro and in vivo experimentations have aimed at understanding the molecular pathways of circRNAs contribution in the evolution of HCC. CircRNAs have interplay with a number of transcription factors such as ZEB1 that possibly mediates the effects of these transcripts in the epithelial-mesenchymal transition. Moreover, circRNAs functionally interact with miRNAs. CircRNA&#95;0000502/ miR-124, circ&#95;0001955/ miR-145-5p, circ&#95;0001955/ miR-516a-5p and hsa&#95;circ&#95;0001955/miR-145-5p are examples of such interactions in the context of HCC. CircRNAs not only predict the course of HCC, but also, they can differentiate HCC samples from non-malignant liver tissues. In this review article, we have provided an inclusive summary of researches that quantified circRNAs profile in HCC. We also provide evidence for application of circRNAs as HCC biomarkers., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Circular RNAs (cirRNAs) are generally considered as non-coding RNAs which can act as molecular sponges for miRNAs, exert regulatory roles in transcription or splicing, and interplay with RNA binding proteins. These single-stranded transcripts can affect tumor growth, the metastatic ability of cancer cells, stemness properties, and resistance to therapeutic options. Recent investigations have shown the crucial effects of circrNAs in the evolution of breast cancer. Signature of circRNAs in breast cancer samples has been mostly assessed through microarray-based methods revealing up-regulation of some circRNAs such as circ-TFF1, circACAP2, circ-TFCP2L1, hsa&#95;circ&#95;0000519, circDENND4C, circPLK1 and circRNA&#95;069718, while down-regulation of other circRNAs such as hsa&#95;circ&#95;0000375, circYap, hsa&#95;circ&#95;0025202, circTADA2A-E6, circASS1 and circRNA&#95;BARD1 in breast cancer samples. Mechanistically, these transcripts mainly affect breast cancer tumorigenesis via serving as sponges for miRNAs. In the current manuscript, we explore the results of researches that appraised the role of circRNAs in breast cancer., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Brain cytoplasmic 200 (BC200) or alternatively named as brain cytoplasmic RNA 1 (BCYRN1) is a long non-coding RNA (lncRNA) primarily identified in the neurons. In addition to its participation in the pathogenesis of neurodegenerative disorders, it partake in the carcinogenesis process. Numerous in vitro studies have reported elevation of expression of BCYRN1 in cancer cell lines. Short hairpin-RNA-mediated silencing of BCYRN1 has attenuated growth of tumors in the animal models. Independent studies in esophageal squamous cell cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma and non-small cell lung cancer have demonstrated association between elevated BCYRN1 levels and poor survival of patients. Taken together, BCYRN1 is an appropriate candidate for targeted therapies in the field of cancer., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Liver cancer is the 7 th utmost frequent neoplasm and the 4 th principal source of cancer deaths. This malignancy is linked with several environmental and lifestyle-related factors emphasizing the role of epigenetics in its pathogenesis. MicroRNAs (miRNAs) have been regarded as potent epigenetic mechanisms partaking in the pathogenesis of liver cancer. Dysregulation of miRNAs has been related with poor outcome of patients with liver cancer. In the current manuscript, we provide a concise review of the results of recent studies about the role of miRNAs in the progression of liver cancer and their diagnostic and prognostic utility., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Growth arrest-specific gene 6 (GAS6) is a growth factor-like cytokine whose function is related with vitamin K. This protein interacts with receptor tyrosine kinase proteins such as Tyro3, Axl, and TAM Receptor family, therefore affecting the tumorigenic processes via different mechanisms. GAS6-antisense 1 (GAS6-AS1) is a long non-coding RNAs (lncRNAs) that is transcribed from a genomic regions nearby GAS6. This lncRNA is also implicated in the pathobiology of cancer. We intended to judge the role of GAS6 and GAS6-AS1 in the pathogenesis of breast cancer through appraisal of their expression levels in breast cancer tissues and their paired neighboring non-cancerous samples. Expression of GAS6 was up-regulated in breast cancer tissues compared with neighboring tissues (Ratio of Mean Expressions = 2.18, P value = 4.98E-02). On the other hand, expression of GAS6-AS1 was down-regulated in breast tumor tissues compared with controls (Ratio of Mean Expressions = 0.37, P value = 4.26E-03). There were substantial correlations between expression levels GAS6 and GAS6-AS1 in non-cancerous tissues (r = 0.74, P value = 1.47e-13) and cancer tissues (r = 0.85, P value = 2.28e-20). Expression of GAS6-AS was associated with progesterone receptor status (P value = 1.36E-02). However, expressions of this gene and the sense transcript were not linked with any other clinical or demographic variable. Taken together, GAS6 and GAS6-AS1 might partake in the development of breast cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: MicroRNAs (miRNAs) play an essential role in the susceptibility and development of cancer cells., Objective: Examining the dependency of breast cancer risk with genetic polymorphisms of miR-1307, miR-1269, and miR-3117 in a sample of Iranian women (southeast region)., Methods: The case-control study consisted of 520 individuals (260 diagnosed BC patients, 260 healthy individuals). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for genotyping of miR-1307 rs7911488, miR-1269 rs73239138, and miR-3117 (rs4655646 and rs7512692) polymorphisms., Results and Conclusion: This study provided evidence that miR-1307 rs7911488 polymorphism significantly reduced the risk of BC in heterozygous AG genotype, as well as dominant (AG+GG) genotype and G allele. A significant correlation was found between dominant (AA+AG) genotype, the A allele and protection against BC due to miR-1269 rs73239138 in the sample of study. In contrast, our findings suggested that AG genotype and G allele of miR-3117 rs4655646 polymorphism could increase BC's susceptibility among the southeastern Iranian females. The miR-3117 rs7512692 variant also increased the risk of BC in codominant, dominant and recessive models, as well as the T allele. The possible dependency of miR-1307, miR-1269, and miR-3117 variants with patients' clinicopathological characteristics and BC was also studied. It was concluded that there is a correlation between miR-3117 rs7512692 variant and tumor grade (p=0.031); also, a correlation between miR-1269 rs73239138 variant and progesterone receptor status (p=0.006). The current investigation revealed that miR-1307, miR-1269, and miR-3117 polymorphisms might play a crucial role in the Iranian population's vulnerability to BC., .

Deleted in lymphocytic leukemia 2" (DLEU2) is a long non-coding transcript with several splice variants. It has been identified through a comprehensive sequencing of a commonly deleted region in leukemia i.e. the 13q14 region. Afterwards, different investigations reported up-regulation of this long non-coding RNA in several types of cancers. Up-regulation of DLEU2 has been shown to determine poor survival in esophageal, pancreatic, laryngeal, renal, cervical and lung cancers. However, the diagnostic power of DLUE2 has only assessed in two studies; only one them exhibiting promising results. A limitation of most of studies is that they did not differentiate between transcript variants of this lncRNA. Therefore, it is not possible to attribute the observed functions to a certain alternate transcript. In this manuscript, we discuss the results of these studies in three different sections based on the type of experiments., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Esophageal cancer is the seventh most common cancer worldwide. Although a number of environmental and lifestyle-related risk factors have been identified for this kind of cancer, the exact molecular mechanisms of tumor evolution have not been clarified yet. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) as important regulators of gene expression and chromatin configuration have essential roles in the pathogenesis of esophageal cancer. They have been shown to alter the function of cancer-related signaling pathways such as phosphoinositide 3-kinase/protein kinase B and Wnt pathway, thus they might modulate the response of patients to pathway-targeted therapies. Moreover, a number of lncRNAs, such as AFAP1-AS1, UCA1, HOTAIR, LOC285194, and TUSC7, are involved in conferring chemoresistant/radioresistant in esophageal cancer cells. A complex network of interaction exists between lncRNAs and miRNAs in the context of esophageal cancer. Finally, various panels of lncRNAs and miRNAs have been introduced that can predict the survival of esophageal cancer patients. In this review article, we summarize the recent findings regarding the role of miRNAs and lncRNAs in the pathogenesis of esophageal cancer with the special focus on their regulatory roles on signaling pathways, their potential as diagnostic/prognostic markers, and their relevance with therapeutic response., (© 2020 Wiley Periodicals LLC.)

MicroRNAs (miRNAs) are transcripts with sizes of about 22 nucleotides, which are produced through a multistep process in the nucleus and cytoplasm. These transcripts modulate the expression of their target genes through binding with certain target regions, particularly 3' suntranslated regions. They are involved in the pathogenesis of several kinds of cancers, such as glioblastoma. Several miRNAs, including miR-10b, miR-21, miR-17-92-cluster, and miR-93, have been up-regulated in glioblastoma cell lines and clinical samples. On the other hand, expression of miR-7, miR-29b, miR-32, miR-34, miR-181 family members, and a number of other miRNAs have been decreased in this type of cancer. In the current review, we explain the role of miRNAs in the pathogenesis of glioblastoma through providing a summary of studies that reported dysregulation of these epigenetic effectors in this kind of brain cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Prostate cancer is the most common cancer in males in several regions. One of the major challenges in diagnosis and treatment of this cancer is how to identify men who harbor an increased risk of having clinically significant prostate cancer and how to assess response to therapy. Biomarkers, like microRNAs (miRNAs) are one of the new diagnostic/therapeutic tools for clinicians. Finding men at high risk of significant cancer is essential as they will mostly benefit from earlier diagnosis and treatment. At the same time, it is important to reduce the number of unnecessary invasive biopsies in men without (clinically significant) cancer and miRNAs have especial application in this regard. MiRNAs can regulate expression of several genes. Up to 30 percent of protein coding genes are regulated by miRNAs. Based on this critical regulatory role, miRNAs impact cell differentiation, growth and apoptosis. Several studies have reported aberrant expression of miRNAs in different cancers including prostate cancer. miRNAs are regarded as biomarkers in this kind of cancer. Moreover, expression profiles of miRNAs can predict therapeutic response to a number of drugs such docetaxel and some natural agents such as isoflavone. Functional studies have shown that miRNAs regulate a number of critical targets such as Wnt/β-catenin, PI3K/AKT, cyclin dependent kinases, VEGF and JAK/ STAT. Therefore, several aspects of prostate cancer development are influenced by miRNAs. Finally, circulating miRNAs are promising tools for assessment of prostate cancer course and prognosis. In the current review, we summarize the results of studies which reported abnormal expression of miRNAs in prostate cancer and their role as biomarkers or therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are a group of non-coding RNAs that have critical roles in regulation of expression of genes. They can inhibit or decrease expression of target genes mostly via interaction with 3' untranslated region of their targets. Their crucial roles in the regulation of expression of tumor suppressor genes and oncogenes have potentiated them as contributors in tumorigenesis. Moreover, their stability in body fluids has enhanced their potential as cancer biomarkers. In the present review article, we describe the role of miRNAs in the pathogenesis of gastric cancer and advances in application of miRNAs as biomarkers and therapeutic targets in this kind of malignancy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a critical role as a  regulator of immune-system cells, including T cell, natural killer T (NKT), monocytes, dendritic cells (DC), and B cells., Objective: This study aimed to find a possible association between PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) variants and Breast Cancer (BC) risk in a sample of southeast Iranian women., Method: The case-control study consisted of 520 individuals, including 260 histologically confirmed BC patients and 260 non-cancer age-matching healthy women as the control group. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) methods were used for genotyping of PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) polymorphisms., Results and Conclusion: Our findings indicated that the PD-L1 rs4143815 (G/C) variant meaningfully reduced the risk of BC. However, the PD-L1 rs2890658 variant increased the BC risk in the AC genotype as well as the A allele. Furthermore, we could not find a meaningful association between PD-1 rs11568821, PD-1 rs2227981, PD-1 rs2227982, and BC. Our team examined the possible association between variants and clinicopathological characteristics, including age, size of tumour, lymph node, histology, grade of tumour, estrogen and progesterone receptors status as well as human growth factor receptor 2 (HER2). Our findings demonstrated that PD-L1 rs4143815, PD-L1 rs2890658, PD-1 rs2227982 had a significant association with age. Additionally, we found a significant relation between PD-1 rs2227982 variant and tumour size. Statistical analyzes of PD-1 rs2227981 and PD-1 rs11568821 variants showed a meaningful relation between tumour grade and tumour stage (p=0.006), respectively., .

HOXA transcript at the distal tip (HOTTIP) is a 3764 nucleotide long non-coding RNA (lncRNA) encoded from a genomic region in the 5' tip of the HOXA locus. This lncRNA has a role in transmission of signals from higher order chromosomal configuration into chromatin codes. HOTTIP directly binds with the WDR5 protein and recruits the WDR5/MLL complexes across the HOXA locus which leads to H3K4 methylation and activation of the transcription of HOXA genes. This lncRNA has a prominent role in the pathogenesis of almost all kinds of cancers. Apart from a single study in glioma cells, all in vitro studies have emphasized on oncogenic roles of HOTTIP in different malignancies. In vivo studies also showed the effect of HOTTIP silencing in reduction of tumorigenicity in all cancer types except from glioma. Results of clinical studies mostly demonstrated up-regulation of this lncRNA in cancer samples compared with non-malignant tissues of the same origin and correlation between its expression levels and patients' outcome. Taken together, HOTTIP is regarded as an oncogenic lncRNA in almost all kinds of cancers and a putative biomarker and therapeutic target in human malignancies., Competing Interests: Declaration of Competing Interest The authors declare they have no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

The critical roles of long non-coding RNAs (lncRNAs) in the regulation of diverse biological functions has potentiated them as cancer biomarkers. Among these transcripts is the prostate cancer associated transcript 1 (PCAT1) which has been initially shown to exert oncogenic roles in prostate cancer. Further studies revealed its similar roles in various kinds of human malignancies including both solid tumors and hematological malignancies. Animal studies have shown that down-regulation of this lncRNA can attenuate tumor growth in a wide array of cancers including prostate cancer, colorectal cancer, squamous cell carcinoma lung cancer and hepatocellular carcinoma. Studies aimed at identification of diagnostic value of this lncRNA in human cancers reported various values ranging from 0.66 to 0.89 in diverse cancers with the best value reported in multiple myeloma. This lncRNA has a number of putative functional genomic variants such as rs1902432, rs2632159, rs1026411, rs710886, rs16901904 and rs710886 which can modify expression or function of PCAT1 thus altering the risk of human cancers. Based on aberrant expression of PCAT1 in malignancies of diverse origins, this lncRNA can be regarded as a therapeutic target in a vast array of cancers. Thus, modalities for efficient reduction of its expression would be beneficial for several patients., Competing Interests: Declaration of Competing Interest The authors declare they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

No longer regarded as junk DNA, long non-coding RNAs (lncRNAs) are considered as master regulators of cancer development and metastasis nowadays. Among the recently appreciated roles of these transcripts is their fundamental contribution in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Notably, lncRNAs may have interactions with some environmental risk factors for this type of cancer. Moreover, a number of studies have verified diagnostic and prognostic values of lncRNAs in HNSCC. Emerging evidences from functional studies point to the possibility of design of lncRNA-targeted therapies in HNSCC. In the current review, we discuss the molecular mechanisms for participation of lncRNAs in the pathogenesis of HNSCC, their potential application in cancer diagnosis and most importantly in the development of personalized methods for treatment of HNSCC., Competing Interests: Declaration of Competing Interest The authors declare they have nothing to report., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The nuclear factor-κB (NF-κB) has a prominent role in development of breast cancer and response of patients to conventional therapies. Several factors regulate the activity of this transcription factor. In the current investigation, we compared expression levels of five long non-coding RNAs (lncRNAs) with putative interactions with NF-κB namely CHAST, ADINR, DICER1-AS1, HNF1A-AS1 and NKILA between 78 breast cancer tissues and their paired adjacent non-cancerous tissues (ANCTs). We also assessed expression levels of ATG5 and CEBPA mRNA coding genes that are functionally linked with NF-κB signaling in these two sets of samples. All assessed genes except for NKILA were significantly down-regulated in tumoral tissues compared with ANCTs. Expression of NKILA was not significantly different between tumoral tissues and ANCTs. Expression levels of CEBPA and HNF1A-AS were significantly associated with cancer stage (P values of 0.03 and 0.02 respectively). Expression levels of ATG5 tended to be associated with mitotic rate (P = .05). The association between expression levels of ATG5 and tumor size was also significant (P = .02). Expression of CHAST was significantly associated with PR status (P = .04) and tended to be associated with ER status (P = .05). Finally, expression of NKILA was significantly associated with first pregnancy age (P = .01). No other significant association was detected between expression levels of assessed genes and clinical parameters. Expression levels of mentioned genes were significantly correlated with each other. The most significant correlations were found between CHAST and ADINR (correlation coefficients of 0.78 and 0.69 in tumoral tissues and ANCTs respectively). Based on the area under curve (AUC) values, DICER1-AS and CEBPA had the best performance in differentiation of tumoral tissues from ANCTs (AUC values of 0.92 and 0.90 respectively. Combination of transcript quantities of six genes could differentiate these two sets of samples with 92.3% sensitivity, 91% specificity and diagnostic power of 95%. The current project highlights dysregulation of NF-κB-associated genes in breast cancer tissues and suggests them as potential diagnostic markers in breast cancer., Competing Interests: Declaration of Competing Interest The authors declare they have no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is among long non-coding RNAs (lncRNAs) which has disapproved the old term of "junk DNA" which was used for majority of human genome which are not transcribed to proteins. An extensive portion of literature points to the fundamental role of this lncRNA in tumorigenesis process of diverse cancers ranging from solid tumors to leukemia. Being firstly identified in lung cancer, it has prognostic and diagnostic values in several cancer types. Consistent with the proposed oncogenic roles for this lncRNA, most of studies have shown up-regulation of MALAT1 in malignant tissues compared with non-malignant/normal tissues of the same source. However, few studies have shown down-regulation of MALAT1 in breast cancer, endometrial cancer, colorectal cancer and glioma. In the current study, we have conducted a comprehensive literature search and provided an up-date on the role of MALAT1 in cancer biology. Our investigation underscores a potential role as a diagnostic/prognostic marker and a putative therapeutic target for MALAT1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Long non-coding RNAs (lncRNAs) have fundamental roles in cell migration, proliferation, invasion and metastasis., Methods: In the current study, we evaluated expression of a panel of lncRNAs in bladder cancer tissues, adjacent non-cancerous tissues (ANCTs) and normal bladder tissues to evaluate their diagnostic power., Results: PV1 was down-regulated in tumor tissues compared with both ANCTs and normal controls (Expression ratios of 0.48 and 0.14; P values of 0.4 and <0.001 respectively). HOTAIR, NEAT1, TUG1 and FAS-AS1 were significantly down-regulated in tumor tissues compared with normal controls (Expression ratios of 0.4, 0.68, 0.54 and 0.11; P values of 0.04, 0.02, 0.02 and <0.001 respectively)., Conclusion: Combination of transcript levels of seven lncRNAs improved both sensitivity and specificity values to 100%. The current study shows dysregulation of lncRNAs in bladder cancer and implies their role as diagnostic markers in this malignancy., (Copyright © 2019. Published by Elsevier Inc.)

β-secretase (BACE1) and its naturally occurring anti-sense RNA (BACE1-AS) have established role in the pathologic process leading to Alzheimer's disease. Their possible implication in the neoangiogenesis suggests that they might be involved in the tumorigenesis events as well. In the present study, we compared transcript levels of these genes in 30 gastric cancer samples and their adjacent non-cancerous tissues (ANCTs) to find whether their altered expression might facilitate discrimination of these two sets of samples. Expressions of both genes were associated with site of primary tumor. Both genes were significantly down-regulated in tumoral tissues compared with ANCTs. Significant correlations were detected between transcript levels of these genes in both sets of samples. Transcript levels of BACE1 and BACE1-AS had the diagnostic power of 75% based on Receiver operating characteristic curve analysis. The current study provides evidences for contribution of BACE1 and BACE1-AS in gastric cancer evolution and suggests their potential as diagnostic markers.

Aim: Nicotinamide Nucleotide Transhydrogenase (NNT) gene encodes a protein, which is an important antioxidative enzyme that converts NADH to NADPH. This enzyme provides a significant proportion of the entire NADPH resource in the mitochondria. Previous reports have shown possible contribution of this gene in the carcinogenesis process., Methods: In the current research, we evaluated expression levels of NNT gene and a naturally occurring antisense RNA (NNT-AS1) in gastric cancer specimens compared to their corresponding adjacent noncancerous tissues (ANCTs)., Results: Both NNT1 and NNT-AS1 genes were significantly downregulated in tumor tissues compared to ANCTs (expression ratio = 0.369, p = .045 and expression ratio = 0.368, p = .043, respectively). Transcript levels of NNT1 and NNT-AS1 were associated with the location of the primary tumor (p = .003 and .002, respectively). Moreover, expressions of both genes were significantly elevated in tumors with lymphatic/vascular invasion compared to tumors without lymphatic/vascular invasion (p = .001 and p = .005). No other remarkable associations were noticed between transcript levels of genes in tumor tissues and patients' information. Based on the area under curve (AUC) values in the receiver operating characteristic (ROC) curves, the diagnostic power of NNT1 and NNT-AS1 were estimated to be 0.62 and 0.63, respectively., Conclusions: Although we demonstrated dysregulation of NNT1 and NNT-AS1 in gastric tumor specimens in association with clinical data of patients, these two genes are not supposed to be appropriate biomarkers for gastric cancer., (© 2019 John Wiley & Sons Australia, Ltd.)

Long noncoding RNAs (lncRNAs) have been involved in the pathogenesis of several human cancers including gastric cancer. In the current study, we selected five lncRNAs namely NEAT1, TUG1, PANDA, UCA1, and GHET1 to assess their expressions in gastric cancer samples compared with adjacent noncancerous tissues (ANCTs) from the same patients. Some previous reports have shown contribution of these lncRNAs in gastric cancer. However, we aimed to explore their associations with patients' clinicopathological data and their potential as diagnostic biomarkers. Significant associations were found between site of primary tumor and relative expression of all lncRNAs in cancer samples compared with ANCTs. Besides, GHET1 relative expression was associated with lymph node status. The diagnostic power of GHET1 was higher from other lncRNAs. Combination of GHET1, TUG1, UCA1, and PANDA increased the diagnostic power and significance (AUC = 0.8; P < 0.0001). The current study supports participation of lncRNAs in the pathogenesis of gastric cancer and highlights their potential as diagnostic biomarkers., (© 2019 Wiley Periodicals, Inc.)

Background: Non-small cell lung cancer (NSCLC) as the most frequent type of lung cancer is associated with extensive mortality. Researchers have studied the suitability of several molecules as biomarkers for early detection of this cancer. Long non-coding RNAs (lncRNAs) as the main regulators of gene expression have also been assessed in this regard., Methods: In the present study, we compared expression level of Fas-antisense 1 (FAS-AS1), Growth Arrest Specific 5 (GAS5), PVT1, Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1), taurine upregulated gene 1 (TUG1) and TNFα and hnRNPL related immunoregulatory LincRNA (THRIL) in 32 NSCLC samples and their corresponding adjacent non-cancerous tissues (ANCTs)., Results: NEAT1 has been significantly over-expressed in NSCLC tissues obtained from male subjects compared with the corresponding ANCTs (Relative expression (REx) = 3.022, P = 0.019) but not in female subjects (P = 0.975). FAS-AS1 was significantly down-regulated in NSCLC tissues obtained from both males and females subjects compared with the corresponding ANCTs (REx = - 4.12 and - 3.14, P = 0.015 and 0.033 respectively). TUG1, GAS5, THRIL and HOTAIRM1 were significantly down-regulated in tumoral tissues obtained from male subjects compared with the corresponding ANCTs., Conclusions: The observed dysregulation of these lncRNAs in NSCLC tissues compared with the corresponding ANCTs warrants future studies to confirm the results of the current study in larger sample sizes to elaborate their role as cancer biomarkers.

Background: The long non-coding RNA (lncRNA) DSCAM-AS1 has been demonstrated to participate in the pathogenesis of breast cancer and tamoxifen resistance., Objective: To evaluate expression profile of DSCAM-AS1 in invasive ductal carcinoma of breast and its suitability as a biomarker for diagnosis of breast cancer., Methods: We evaluated expression of DSCAM-AS1 in 108 breast tissues including tumoral and adjacent non-cancerous tissues (ANCTs) by means of quantitative real time PCR., Results: DSCAM-AS1 was up-regulated in tumoral tissues compared with ANCTs (Fold change = 2.86, P = 0.011). Its expression was significantly higher in patients aged less than 55 compared with older patients (P = 0.02). However, its expression levels had not a good performance as a diagnostic biomarker for breast cancer., Conclusions: The significant up-regulation of DSCAM-AS1 in tumoral tissues compared with ANCTs provides further evidences for participation of this lncRNA in the pathogenesis of breast cancer.

Background: Breast cancer is the most common malignancy in women. Multidrug resistance (MDR) is still a great obstacle of breast cancer chemotherapy. We have previously shown that multidrug resistance-associated protein 1 (MRP1) is associated with response to neoadjuvant chemotherapy. The lung resistance-related protein (LRP) is identified as a prognostic marker and response to treatment factor which has been studied mainly in hematological malignancy and leukemia. In this study, we aimed to analyze LRP expression and possible correlation between the expression level of this gene with MRP1 as a candidate marker for chemotherapy resistance. Materials and Methods: We collected 54 breast tumors and adjacent normal tissues from Iranian breast cancer patients and Real time RT-PCR was employed to measure the gene expression level in our samples. Results: MRP1 and LRP expression level were significantly lower in tumor tissues of the patients responding to chemotherapy compared to non-responding patients. No relation between the expression level of either of these genes and clinicopathology markers was found. Conclusion: Our results suggest that LRP gene expression is correlated to MRP1 in human breast cancer cells and may affect the clinical response to treatment., (Creative Commons Attribution License)

Background: The suppressor of cytokine signaling (SOCS) family of proteins are inhibitors of the cytokine-activated Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. We aimed at evaluation of expression of SOCS genes in breast cancer., Methods: We evaluated expression of SOCS1-3 and SOCS5 genes in breast cancer samples compared with the corresponding adjacent non-cancerous tissues (ANCTs)., Results: All assessed SOCS genes were significantly downregulated in tumoral tissues compared with ANCTs. SOCS1 and SOCS2 genes were significantly overexpressed in higher grade samples, but SOCS3 had the opposite trend. Significant correlations were found between expression levels of SOCS genes. The SOCS1 and SOCS2 expression levels had the best specificity and sensitivity values respectively for breast cancer diagnosis., Conclusion: The current study provides further evidence for contribution of SOCS genes in breast cancer.

Background: The role of long non-coding RNAs has been extensively appreciated in the contexts of cancer. Interferon γ-antisense RNA1 (IFNG-AS1) is an lncRNA located near to IFN-γ-encoding (IFNG) gene and regulates expression of IFNG in Th1 cells., Methods: In the present study, we evaluated expression of IFNG and IFNG-AS1 in 108 breast samples including tumoral tissues and their adjacent non-cancerous tissues (ANCTs) using real-time PCR. IFNG-AS1 was significantly upregulated in tumoral tissues compared with ANCTs (expression ratio = 2.23, P = 0.03)., Results: Although the expression of IFNG was higher in tumoral tissues compared with ANCTs (relative expression = 1.89), it did not reach the level of significance (P = 0.07). IFNG expression was significantly higher in HER2-negative tumoral tissues compared with HER2-positive ones (P = 0.01) and in grade 1 samples compared with grade 2 ones (P = 0.03). No other significant difference was found in expressions of genes between other groups., Conclusion: Significant strong correlations were detected between expression of IFNG and IFNG-AS1 in both tumoral tissues and ANCTs. The present study provides evidences for participation of IFNG and IFNG-AS1 in the pathogenesis of breast cancer and warrants future studies to elaborate the underlying mechanism.

Background: Telomeres are involved in chromosomal stability, cellular immortality and tumorigenesis. Human telomerase reverse transcriptase (TERT) is essential for the maintenance of telomere DNA length. Recently, a variable tandem-repeats polymorphism, MNS16A, located in the downstream region of the TERT gene, was reported to have an effect on TERT expression and telomerase activity. Previous studies have linked both relative telomere length (RTL) and TERT variants with cancer. Therefore, we evaluated associations between RTL, TERT gene polymorphisms (hTERT, rs2735940 C/T and MNS16A Ins/Del) and risk of childhood acute lymphoblastic leukemia (ALL) in an Iranian population. Methods: RTL was determined by a multiplex quantitative PCR-based method, and variants of the hTERT, rs2735940 C/T and MNS16A Ins/Del, were genotyped by amplification refractory mutation system PCR (ARMS-PCR), and PCR, respectively. Results: Our results indicated that RTL was shorter in ALL patients (1.53±0.12) compared to the control group (2.04±0.19) (P=0.029). However, no associations between hTERT gene variants or haplotypes and the risk of childhood ALL were observed (P>0.05). Also hTERT polymorphisms were not associated with RTL or patient clinicopathological characteristics, including age (P=0.304), sex (P=0.061) organomegally (P=0.212) CSF involvement (P=0.966) or response to treatment (P=0.58). Conclusions: We found that telomere attrition may be related to the pathogenesis of childhood ALL, irrespective to TERT variants., (Creative Commons Attribution License)

Background: Lung cancer as the most fatal cancer of men has prompted researchers to find biomarkers for early detection and prognosis. Among the possible biomarkers are a group of non-coding transcripts with sizes more than 200 nucleotides called long non-coding RNAs (lncRNAs)., Aims: In the present study, we evaluated the expression levels of the lncRNA OIP5 antisense RNA 1 (OIP5-AS1) in 32 non-small cell lung cancer (NSCLC) samples compared with their corresponding adjacent non-cancerous tissue (ANCTs) by means of real-time polymerase chain reaction. The samples were obtained from patients who were admitted at Labbafi-Nejad Hospital during 2015 and 2016., Results: OIP5-AS expression levels was significantly decreased in tumoral tissues compared with ANCTs in total samples and in male subgroup. However, no association was found between relative expression of OIP5-AS1 and clinicopathological data of patients or history of smoking. Expression levels of this lncRNA were not correlated with patients age., Conclusions: This lncRNA is possibly a novel biomarker of NSCLC in Iranian patients. Future studies are needed to confirm the results of our study in larger sample sizes. Moreover, based on the difference in lung cancer associated risk factors in different populations, population-based studies are needed to explore the role of this lncRNA in the pathogenesis of cancers in each region to design appropriate targeted therapies for each population. Key words: lung cancer - OIP5-AS - lncRNA - long non-coding RNA.

Background: Fibulin-4 (FBLN-4) is an extracellular glycoprotein that is upregulated in some cancer and is khown as prognostic marker in ovarian and cervical cancer. Breast cancer resistance protein (BCRP) is an ATP-binding cassette transporter that facilitates the efflux of various anticancer drugs from the cell and cause MDR phenotype in breast tumors. Many studies are available that indicat overexpression of BCRP gene in breast cancer., Objective: In the present study we aimed to analyze the expression level of FBLN-4 and BCRP in Iranian breast cancer patients., Methods: We collected 40 samples of breast cancer and normal tissue from Tehran Khatam-al-Anbia hospital. To analyze the gene expression by using Real Time RT-PCR FBLN-4 and BCRP gene expression level were measured and then the association of gene expression with breast cancer were determined., Results: Surprisingly the expression level of FBLN-4 and BCRP genes were downregulated in tumor tissues compared to adjacent normal tissues. Comparison of the gene expression and clinico-pathology reports indicate FBLN-4 gene expression was associated with breast cancer histological grade. We found no correlation between the expressions of BCRP gene with any clinico-pathological characters., Conclusion: Interestingly and in contrast with our expectation, we found that the expression level of FBLN-4 and BCRP were downregulated in tumor compared to adjacent normal tissues. FBLN-4 was associated with grade histology and therefore can be considered as a potential prognostic biomarker.

The membrane epithelial mucin MUC1 is expressed at the luminal surface of most simple epithelial cells, but expression is greatly increased in most breast cancers. The aims of present study were to investigate expression of the MUC1 gene and interactive affects in metastases. Whole cell RNA isolation from 50 sentinel lymph nodes (SNLs) of breast cancer patients was performed using reverse transcription and real-time PCR. All patients were diagnosed with breast cancer and without metastasis, confirmed by IHC staining. The evaluation of tumor and normal samples for expression of MUC1 gene, the results were 49.1% non-expressive and 45.3% expression (Student t, p = 0.03). Also in comparison of normal breast tissue and breast cancer SLN for MUC1 gene, MUC1 negative SLNs were 75.0% (18 samples) and MUC1 positive samples were 25.0% (6 samples). Over-expression of MUC1 gene may offer a target for therapy related to progression and metastasis in women with breast cancer.

Breast cancer is the most prevalent type of cancer among women around the world, and mortality is primarily caused by micro-metastatic disease. The complex mechanisms of breast cancer invasion and metastasis are intrinsically related to the malignant cell type so that early detection of micro-metastases can help prolongation of survival for patient. The aim of the present research work was evaluation of the expression status of mammoglobin protein as a candidate molecular marker in the negative sentinel lymph node (SLN). Fifty tumor specimens, and 50 normal adjacent breast tissue samples from the same patients were selected on the basis of having more than 10% tumor content for RNA extraction from SLNs. Tumor samples and normal adjacent breast tissue were archived in the form of frozen fresh tissue in liquid nitrogen. Real-time PCR was performed on a Bioner life express gradient thermal cycler system. Mammoglobin gene overexpression in breast cancer metastasis was investigated. Single marker results were mammaglobin 66.7% and CK19 50.0%, with 58.3% for the two in combination. Due to improved outcome with at least 3 genes (83.3%), it seems, triple marker evaluation will be most likely useful for detecting micro-metastases instead of studying separate genes.

Detection of micrometastasis in sentinel lymph nodes (SLNs) is a very useful tool for appropriate assessment of the clinical stage of disease in breast cancer patients. Early identification of clinically relevant disease could lead to early treatment or staging approaches for breast cancer patient. Micrometastases in SLNs of women with invasive breast cancer are of great significance in this context. In this study we examined SLN biopsies considered to have small numbers of cancerous cells by real time RT-PCR. All of the samples underwent immunohistochemical staining for cytokeratin for confirmation of the presence or absence of micrometastases. BUB1b expression assay of selected patients with and without metastasis showed overexpression in the former, but not in normal breast and lymph node tissue. Our results may be taken into account in the discussion about the merits of routine use of molecular assessment in pathogenetic studies of SLNs.

The transitional endoplasmic reticulum (tER) is composed of both rough and smooth ER membranes and thus participates in functions attributed to both these two subcellular compartments. In this paper we have compared the protein composition of tER isolated from dissected liver tumor nodules of aflatoxin B1-treated rats with that of tER from control liver. Tandem mass spectrometry (MS), peptide counts and immunoblot validation were used to identify and determine the relative expression level of proteins. Inhibitors of apoptosis (i.e. PGRMC1, tripeptidyl peptidase II), proteins involved in ribosome biogenesis (i.e. nucleophosmin, nucleolin), proteins involved in translation (i.e. eEF-2, and subunits of eIF-3), proteins involved in ubiquitin metabolism (i.e. proteasome subunits, USP10) and proteins involved in membrane traffic (i.e. SEC13-like 1, SEC23B, dynactin 1) were found overexpressed in tumor tER. Transcription factors (i.e. Pur-beta, BTF3) and molecular targets for C-Myc and NF-kappa B were observed overexpressed in tER from tumor nodules. Down-regulated proteins included cytochrome P450 proteins and enzymes involved in fatty acid metabolism and in steroid metabolism. Unexpectedly expression of the protein folding machinery (i.e. calreticulin) and proteins of the MHC class I peptide-loading complex did not change. Proteins of unknown function were detected in association with the tER and the novel proteins showing differential expression are potential new tumor markers. In many cases differential expression of proteins in tumor tER was comparable to that of corresponding genes reported in the Oncomine human database. Thus the molecular profile of tumor tER is different and this may confer survival advantage to tumor cells in cancer., (Copyright © 2010 Elsevier B.V. All rights reserved.)