RET Gene Expression Research Articles

RET enhancer haplotype-dependent remodeling of the human fetal gut development program.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.

Targeted therapy of RET fusion-positive non-small cell lung cancer.

Lung cancer has very high morbidity and mortality worldwide, and the prognosis is not optimistic. Previous treatments for non-small cell lung cancer (NSCLC) have limited efficacy, and targeted drugs for some gene mutations have been used in NSCLC with considerable efficacy. The RET proto-oncogene is located on the long arm of chromosome 10 with a length of 60,000 bp, and the expression of RET gene affects cell survival, proliferation, growth and differentiation. This review will describe the basic characteristics and common fusion methods of RET genes; analyze the advantages and disadvantages of different RET fusion detection methods; summarize and discuss the recent application of non-selective and selective RET fusion-positive inhibitors, such as Vandetanib, Selpercatinib, Pralsetinib and Alectinib; discuss the mechanism and coping strategies of resistance to RET fusion-positive inhibitors.

Correlation analysis of clock genes and MEN2 medullary thyroid carcinoma

Objective: To investigate the correlation between CLOCK and BMAL1 genes and MEN2 medullary thyroid carcinoma (MTC). Methods: Thirteen cases with MEN2 MTC and thirteen cases with non-MEN2 MTC were selected who were treated in the Yantai Yuhuangding Hospital between January 2013 and September 2021. Clinical indicators such as blood calcitonin level, tumor diameter and metastatic lymph node of patients were collected. The expression differences of CLOCK and BMAL1 between MEN2 MTC and para-carcinoma tissue as well as between MEN2 MTC and non-MEN2 MTC were detected by immunohistochemistry and qPCR. The correlation between lymph node metastasis and CLOCK or BMAL1 expression was analyzed. Protein-protein interaction (PPI) network analysis combined with qPCR and correlation analysis was used to explore the expression regulation relationship between RET and circadian clock genes. The rhythm disorder of MEN2 cells was verified by lipopolysaccharide cell stimulation experiment after dexamethasone rhythm synchronization. Results: MEN2 MTC exhibited typical RET gene mutation. The mean blood calcitonin level, the tumor diameter and the number of metastatic lymph nodes of patients with MEN2 MTC were higher than those of patients with non-MEN2 MTC (t value was 2.76, 2.53, 2.26, all P<0.05). Immunohistochemical results showed that the expression levels of CLOCK and BMAL1 in MEN2 MTC were higher than those in non-MEN2 MTC, while negatively expressed in para-cancerous thyroid follicle. qPCR displayed that the expression of CLOCK gene in cancer tissues was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.68 and 2.86, all P<0.05); the expression of BMAL1 gene in MEN2 MTC was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.21 and 2.35, all P<0.05). Correlation analysis showed that the expression levels of CLOCK and BMAL1 genes were positively correlated with the number of lymph node metastases in patients with MEN2 MTC (r=0.65, P<0.001; r=0.52, P=0.005). PPI network analysis indicated that the expression of CLOCK gene was positively correlated with the abnormal expression of RET gene (r=0.96, P<0.001). With lipopolysaccharide to stimulate cultured cells in vitro after dexamethasone rhythm synchronization, the expressions of CLOCK and BMAL1 in MEN2 MTC cells (0.47±0.22 and 2.60±1.48) at 12 hours of synchronization were significantly lower than those in para-cancerous tissues (1.70±1.62 and 8.23±2.52), the difference was statistically significant(t=5.04, P=0.007; t=3.34, P=0.029). Conclusion: CLOCK and BMAL1 are correlated with the occurrence and development of MEN2 MTC, and may be potential targets for the development of new therapeutic strategies for MEN2 MTC.

CRISPR/Cas9 RET Gene Knockout in Medullary Thyroid Carcinoma Cell-lines: Optimization and Validation.

Medullary Thyroid Cancer (MTC) is a very aggressive type of thyroid carcinoma. Mutation in RET proto-oncogene is demonstrated in MTC development. We aimed to knock-out of RET-oncogene using CRISPR/Cas9 genome editing method in MTC cell-lines. This research was conducted in Shahid Beheshti University of Medical Sciences, Tehran, Iran during 2019-2020. Four different sgRNAs were designed to target exons one, two, and four of RET-oncogene in TT and MZ-CRC-1 cell-lines using bioinformatics tools, then the CRISPR/Cas9 constructs was made. About 72-hours after cell transfection, T7EI method and DNA sequencing were used to confirm the knock-out of RET-oncogene. Expression of RET, Calcitonin genes and RET protein were evaluated by Real-time PCR and ELISA, respectively. The results of T7E1, and DNA sequencing of transfected cells confirmed RET gene knock-out by CRISPR/Cas9. There was a significant decrease in RET gene expression and RET protein in transfected TT and MZ cells compared to controls. The rate of cell apoptosis in transfected cells was significantly increased. Calcitonin gene expression was also significantly reduced in transfected cells. p-RET, p-PI3K, p-AKT, p-MEK, p-ERK protein levels were significantly reduced in TT and MZ transfected cells. For the first time, knock-out of RET gene was performed and confirmed using CRISPR/Cas9. Inhibition of this gene leads to inhibition of the tyrosine kinase RET signal transduction pathway. Therefore, it can be one of the most effective and specific therapeutic goals in the field of Personalized Medicine in the treatment of diseases caused by over activity of RET molecular pathway.

A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease.

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET–EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET–EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.

Mechanism of RET gene mediated EGFR signaling pathway on epithelial-mesenchymal transition, proliferation and apoptosis of papillary thyroid carcinoma cells.

To explore the mechanism of RET gene mediated EGFR signaling pathway on the epithelial-mesenchymal transition (EMT), proliferation and apoptosis of papillary thyroid carcinoma (PTC) cells. PTC TPC-1 cells and human normal thyroid follicular epithelial cells Nthy-ori 3-1 were collected to identify the expression of RET in PTC. Seven groups were divided according to different transfection protocols, including blank group, negative control group, si-RET group, oe-RET group, AG-490 group, NSC 228155 group, and si-RET + NSC 228155 group. After transfection, qRT-PCR was used to identify whether the transfection was successful or not. qRT-PCR and Western blot were performed to detect the mRNA and protein expressions of RET, EGFR signaling pathway related genes, and EMT related genes. Cell migration, invasion, proliferation and apoptosis abilities were further detected by CCK8, cell scratch, transwell and flow cytometry assays, respectively. RET gene was highly expressed in PTC cells (p<0.05). Compared with blank group, oe-RET group and NSC 228155 group had activated EGFR signaling pathway manifesting in the increased expression of EGFR, p-Src, p-FAK, accelerated EMT showing in the increased expression of N-cadherin and Vimentin expression, but decreased E-cadherin expression, increased cell migration, invasion and proliferation, while decreased apoptosis (all p<0.05); si-RET group and AG-490 group had inhibited activation of EGFR signaling pathway, suppressed EMT, decreased cell migration, invasion and proliferation, while increased apoptosis (all p<0.05); while no evident difference was found in si-RET + NSC 228155 group (all p>0.05). Meanwhile, compared with si-RET group, si-RET + NSC 228155 group showed activated EGFR signaling pathway, accelerated EMT, increased abilities of cell migration, invasion and proliferation, while decreased apoptosis (all p<0.05). RET gene is highly expressed in PTC acting as an oncogene. Silencing RET gene expression may inhibit the invasion and promote the apoptosis of PTC cells by inhibiting the activation of EGFR signaling pathway and mediating the process of EMT. It suggests that RET may offer the possibility of a promising therapeutic target for the treatment of PTC on the basis of the explored mechanism.

Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules.

Molecular tests have clinical utility for thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology, although their performance requires further improvement. This study evaluated the analytical performance of the newly created ThyroSeq v3 test. ThyroSeq v3 is a DNA- and RNA-based next-generation sequencing assay that analyzes 112 genes for a variety of genetic alterations, including point mutations, insertions/deletions, gene fusions, copy number alterations, and abnormal gene expression, and it uses a genomic classifier (GC) to separate malignant lesions from benign lesions. It was validated in 238 tissue samples and 175 FNA samples with known surgical follow-up. Analytical performance studies were conducted. In the training tissue set of samples, ThyroSeq GC detected more than 100 genetic alterations, including BRAF, RAS, TERT, and DICER1 mutations, NTRK1/3, BRAF, and RET fusions, 22q loss, and gene expression alterations. GC cutoffs were established to distinguish cancer from benign nodules with 93.9% sensitivity, 89.4% specificity, and 92.1% accuracy. This correctly classified most papillary, follicular, and Hurthle cell lesions, medullary thyroid carcinomas, and parathyroid lesions. In the FNA validation set, the GC sensitivity was 98.0%, the specificity was 81.8%, and the accuracy was 90.9%. Analytical accuracy studies demonstrated a minimal required nucleic acid input of 2.5 ng, a 12% minimal acceptable tumor content, and reproducible test results under variable stress conditions. The ThyroSeq v3 GC analyzes 5 different classes of molecular alterations and provides high accuracy for detecting all common types of thyroid cancer and parathyroid lesions. The analytical sensitivity, specificity, and robustness of the test have been successfully validated and indicate its suitability for clinical use. Cancer 2018;124:1682-90. © 2018 American Cancer Society.

Abstract 5227: Molecular profiling in African American NSCLC patients to identify novel potential driver mutations

Abstract African Americans continue to have poorer 5 year survival after a lung cancer diagnosis than whites for reasons that remain to be fully characterized. This disparity remains even with advances in treatments targeting specific driver mutations that have improved outcomes for some patients. Although the relative frequency of these genetic alterations varies in subsets of individuals defined by sex, histologic subtype, smoking history and race, little is known about the occurrence of these mutations in African Americans. In this study, we characterize the spectrum of known driver mutations in 200 African American NSCLC patients and seek to identify novel somatic mutations in this population. Initially, the population was screened using the Sequenom LungCarta panel of 216 mutations in 24 genes known to contain alterations associated with lung cancer, RET and ROS1 fusion gene expression, and amplification of FGFR1. Whole-exome sequencing is being performed on tumors from those patients with no known somatic mutations. Paired normal and tumor DNA/RNA samples are being genotyped to distinguish germline from somatic mutations in both the screening and sequencing phases. Initial screening has been completed on 130 patients, with the remaining 70 underway. The mean age of the patients enrolled is 61.7 years, 58.5% are female, and 8.5% are never smokers. In profiling the first 130 African American patients, we find that only 28% of patients (N = 36) carry a known somatic mutation that is not present in the germline, far lower than the 41% reported in white patients in our previous studies. Of the identified mutations, approximately 23.8% are KRAS and 23.8% are PT53 alterations. The next most frequent somatic alterations are in EGFR (16.7%). The remaining alterations occurred in only 1 or 2 tumors and include EPHA3, ERBB2, FGFR1, MET, NOTCH1, NRAS, NTRK2, PIK3CA, PTEN, RET and STK11. Five percent of patients have tumors with two driver mutations. Half of the tumors carrying 2 driver mutations had concurrent alterations in KRAS and TP53. Patients with known genetic alterations were more likely to be female than patients with no known genetic alterations (p = 0.42), but did not differ in age, smoking status, pack-years, family history of lung cancer, history of COPD, stage at diagnosis or histology. Mutations in EGFR were responsible for the differences by sex. Exome sequencing is complete for the first 48 of the 92 patients with no known somatic mutations. Sequences are being aligned using Novoalign and GATK will be used for variant calling; results will be available soon. Genetic profiling of NSCLC in African Americans has the potential to both identify novel mutations, expanding the list of potential targets for tailored treatments, and aid clinical decision making in African American patients leading to improvements in outcomes. Citation Format: Ann G. Schwartz, Angela S. Wenzlaff, Chrissy M. Lusk, Greg Dyson, Aliccia Bollig-Fischer, Susan Land, Sneh Lata, Michele L. Cote, Gerold Bepler, Shirish M. Gadgeel. Molecular profiling in African American NSCLC patients to identify novel potential driver mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5227.

Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients.

Germline and somatic mutations play a crucial role in breast cancer (BC), driving the initiation, progression, response to therapy and outcome of the disease. Hormonal therapy is limited to patients with tumors expressing steroid hormone receptors, such as estrogen receptor (ER), nevertheless resistance often limits its success.The RET gene is known to be involved in neurocristopathies such as Hirschsprung disease and Multiple Endocrine Neoplasia type 2, in the presence of loss-of-function and gain-of-function mutations, respectively. More recently, RET over-expression has emerged as a new player in ER-positive (ER+) BC, and as a potential target to enhance sensitivity and avoid resistance to tamoxifen therapy.Therefore, targeting the RET pathway may lead to new therapies in ER+ BC. To this end, we have investigated the molecular mechanisms which underlie RET overexpression and its possible modulation in two BC cell lines, MCF7 and T47D, showing different RET expression levels. Moreover, we have carried out a pilot association study in 93 ER+ BC patients. Consistent with the adverse role of RET over-expression in BC, increased overall survival was observed in carriers of the variant allele of SNP rs2435357, a RET polymorphism already known to be associated with reduced RET expression.

A30 - Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients

A30 - Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients

210 Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients

Germline and somatic mutations play a crucial role in breast cancer (BC), driving the initiation, progression, response to therapy and outcome of the disease. Hormonal therapy is limited to patients with tumors expressing steroid hormone receptors, such as estrogen receptor (ER), nevertheless resistance often limits its success. The RET gene is known to be involved in neurocristopathies such as Hirschsprung disease and Multiple Endocrine Neoplasia type 2, in the presence of loss-of-function and gain-of-function mutations, respectively. More recently, RET over-expression has emerged as a new player in ER-positive (ER+) BC, and as a potential target to enhance sensitivity and avoid resistance to tamoxifen therapy. Therefore, targeting the RET pathway may lead to new therapies in ER+ BC. To this end, we have investigated the molecular mechanisms which underlie RET overexpression and its possible modulation in two BC cell lines, MCF7 and T47D, showing different RET expression levels. Moreover, we have carried out a pilot association study in 93 ER+ BC patients. Consistent with the adverse role of RET overexpression in BC, increased overall survival was observed in carriers of the variant allele of SNP rs2435357, a RET polymorphism already known to be associated with reduced RET expression.

Abstract 3575: The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel

Abstract Chromosomal translocations and corresponding gene fusions play an important role in the initiation of tumorigenesis and these processes have been strongly associated with distinct tumor subtypes. The recent association of ALK, ROS and RET fusion transcripts as lung tumor therapy predictive biomarkers has increased the need of a technology that could detect these biomarkers starting from limited amount of material. Life Technologies and OncoNetwork consortium collaborated for the development of a lung fusion panel based on Ion AmpliSeq™ RNA chemistry. The OncoNetwork consortium is comprised of twelve -translational cancer research institutes with many years of experience in adopting the latest molecular techniques for lung therapy research. Material and Methods: Consortia's requirements for the panel development : 1) detect all variants of ALK, ROS1, or RET fusion transcripts described in COSMIC in a single reaction using 10 ng of total RNA 2) Include 5′ and 3′ ALK, ROS1, RET gene expression assays as an indicator of a translocation at this gene. 3) Include endogenous RNA assay controls to determine if the quality of the results could be affected by RNA quality. 4) Provide similar results on archived FFPE samples tested by FISH. Human lung adenocarcinoma cell lines H2228 (EML4-ALK positive), HCC78 (SLC4A2-ROS1 positive ) ,LC-2/ad (CCDC6-RET positive) and Ambion® FirstChoice® Human Brain Reference (HBR) RNA was used as positive and negative control for the study. Formalin fixed paraffin embedded (FFPE) tissue was isolated using different extraction methods. After amplification using Ion AmpliSeq™ RNA chemistry samples were sequenced on the Ion Torrent PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit. The presence of the fusions was confirmed by custom TaqMan® gene expression assays when possible. Preliminary results of the panel on ALK or ROS1 or RET positive cell lines and FFPE archived cancer research samples gave good concordance with FISH results. Expected negative samples were confirmed negative. We found a KIF5B-RET fusion positive sample in a sample not previously tested for RET fusions. Two of the expected positive samples by FISH were found negative due to limited amount to tumor cells present in the sample. Cell line RNA dilutions were performed to determine the panel's limit of detection. We demonstrate a limit of detection of 1 % tumor RNA in the presence of 99 % normal RNA using the panel with 10 ng of RNA extracted by cell lines. Gene expression controls work well as an indicator of the RNA quality and of the translocation presence. The Ion AmpliSeq™ RNA lung cancer fusion panel workflow is easier and faster to perform in comparison to the FISH method. The results obtained to date are highly encouraging for panel to be used in the clinical research setting. More data needs to be analyzed before a final conclusion is made. Citation Format: Susan M. Magdaleno, Angie Cheng, Rosella Petraroli, Orla Sheils, Bastiaan Tops, Delphine Le Corre, Henriette Kurth, Helene Blons, Eliana Amato, Andrea Mafficini, Anna Maria Rachiglio, Anne Reimann, Christoph Noppen, Chrysanthi Ainali, Jin Katayama, Renato Franco, Harriet Feilotter, Jeoffrey Schageman, Ian Cree, Andrew Felton, Jose Luis Costa, Alain Rico, Aldo Scarpa, Jose Carlos Machado, Kazuto Nishio, Nicola Normanno, Marjolijn Ligtenberg, Cecily P. Vaughn, Ludovic Lacroix, Pierre Laurent-Puig. The OncoNetwork Consortium: A global collaborative research study on the development and verification of an Ion AmpliSeq RNA gene lung fusion panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3575. doi:10.1158/1538-7445.AM2014-3575

145IN - New Insights for Sequencing Data in Lung Cancer

ABSTRACT Cases of non-small-cell lung cancer (NSCC) carrying EGFR mutations have been shown to be hyperresponive to EGFR tyrosine kinase inhibtors. Chromosomal translocations and corresponding gene fsions play an important role in initiating tumrigeness. Asociation of ALK, ROS1 and RET fusion tanscripts and its potential as lung tumor pedictive biomakes for respective kinase ihibitors has increased the need of a technlogy that could detect these bimarkers starting from imited amoung of FFPE material for recision medicine with molecular targeted agents. We have set up the MssArray Lung Cancer Panel and Lung Fusion Panel (Sequenom) to analyze the FFPE biopsy samples of lung cancer and examined its feasibiliy. Deep sequencing technologis as well as liquid biopsy are powerful tools for this strategy. The OncoNetwork Consortium and Life Technologies collaborated for the development of lung fusion panel based on Ion AmpliSeqTM RNA chemistry to: 1) Detect all variants of ALK, ROS1, or RET fusion transcripts described in COSMIC in a single reaction using 10 ng of total RNA; 2) Include 5′ and 3′ ALK, ROS1, RET gene expression assays as an indicator of translocation at this gene; 3) Include endogenous RNA assay controls to determine if the quality of the results could be affected by RNA quality;4) Provide similar results on archived FFPE smples tested by FISH. Monitoring of the acquired resistance to EGFR-TKI and ALK inhibitors is another important issue for the development of the next generation therapeutics. Rebiopsy is essential to detect secondary mutations of EGFR (T790M) that causes the resistance to EGFR-TKI. However tissue availability limits the genotyping of EGFR in a clinical setting. We have developed the several sensitive assays (Scorpion-Arms, Cobas(R) EGFR mutation test, SABER and droplet-digital PCR (ddPCR) system e.g.) to detect EGFR mutations and fusion genes from tumor-derived DNA in tissue and blood of lung cancer patients. We have monitored the EGFR mutation status during anti-EGFR treatment. We have now conclded that ddPCR system, e.g., is a suitable technology for liquid biopsies. In collaboration with the West Japan Oncology Group (WJOG), we have established a prescreening system to detect marker-positive population using these deep sequencing technologies and ddPCR for the clinical studies. Disclosure: The author has declared no conflicts of interest.

Expression variability and function of the RET gene in adult peripheral blood mononuclear cells.

RET is a gene playing a key role during embryogenesis and in particular during the enteric nervous system development. High levels of RET gene expression are maintained in different human tissues also in adulthood, although their physiological role remains unclear. In particular, collected evidences of a RET contribution in the development and maintenance of the immune system prompted us to investigate its levels of surface expression on peripheral blood mononuclear cells (PBMCs) from adult healthy donors. Despite variability among samples, RET expression was conserved at similar levels in the different immune cell subsets, with higher correlations in similar lymphocyte populations (i.e. CD4(+) and CD8(+) T cells). Conversely, no correlation was found between the amount of RET receptor, the expression of its putative ligands and co-receptors and the genotypes at the RET locus. Moreover, we investigated the RET-associated inflammatory pathways in PBMCs from healthy donors both in resting conditions and upon glial cell derived neurotrophic factor (GDNF) and GPI-linked co-receptors alpha 1 (GFRα1) mediated RET activation. RET mRNA levels positively correlated with the transcript amount of interleukin-8 (IL-8), a cytokine produced by monocytes and macrophages, though we could not demonstrate its direct effect on RET expression by in vitro experiments on THP1 human monocytic cells. These results imply that RET expression might be influenced by either cis- and/or trans-factors, which together would account for its high variability within the general population, and suggest a putative functional role of the RET gene in modulating immune cell responses during inflammation and carcinogenesis.

Frequency of oncogenic alterations in five driver genes in Chinese female lung adenocarcinoma.

1588 Background: Currently, 5 driver genes (EGFR, K-RAS, B-RAF, ALK, RET) have been widely explored and become new targets of NSCLC, however no report has been found in Chinese female lung adenocarcinoma, who were prone to EGFR mutations and therefore, are the targets for TKIs therapy. Methods: FFPE-tissues from 310 female lung adenocarcinoma adopted in Hunan or Henan province between 2000 and 2012 were investigated. Oncogenic alterations in newly found 5 driver genes were analyzed. ARMS was used to study EGFR, K-RAS and B-RAF mutation. The reverse transcription and real-time PCR were performed to detect either ALK and RET fusions or ALK and RET gene expression. Sequencing was further applied to confirm the subtypes of fusions. Results: Among 310 samples, 149 (48.1%) EGFR mutations, 16 (5.2%) KRAS mutations, 0 (0%) BRAF mutations, 23 (7.4%) ALK fusions and 5 (1.6%) RET fusions were detected. Only EML4-ALK fusion but no other ALK fusions were found. Further research showed that cases (22, 95.6%) with high ALK expression were often accompanied by EML4-ALK fusion and poorly differentiated adenocarcinoma. Two RET fusions, KIF5B-RET and CCDC6-RET were found, with 2 cases and 3 cases, respectively. The ratio of RET/ABL mRNA levels was significantly higher in CCDC6-RET samples with poorly differentiated adenocarcinoma. However, with KIF5B-RET fusion, relatively high RET/ABL mRNA expression was detected in one KIF5B-RET sample with moderately differentiated adenocarcinoma, while no RET expression was detected in other two KIF5B-RET samples with highly differentiated adenocarcinoma. Conclusions: This is the first research about the oncogenic alterations of 5 important driver genes in Chinese female lung adenocarcinoma, as well as the correlation between ALK fusion and ALK expression or between RET fusion and RET expression, thus laying good foundation for understanding the genetic mutation spectrum in female lung adenocarcinoma.

Multiple endocrine neoplasias type 2B and RET proto-oncogene.

Multiple Endocrine Neoplasia type 2B (MEN 2B) is an autosomal dominant complex oncologic neurocristopathy including medullary thyroid carcinoma, pheochromocytoma, gastrointestinal disorders, marphanoid face, and mucosal multiple ganglioneuromas. Medullary thyroid carcinoma is the major cause of mortality in MEN 2B syndrome, and it often appears during the first years of life. RET proto-oncogene germline activating mutations are causative for MEN 2B. The 95% of MEN 2B patients are associated with a point mutation in exon 16 (M918/T). A second point mutation at codon 883 has been found in 2%-3% of MEN 2B cases. RET proto-oncogene is also involved in different neoplastic and not neoplastic neurocristopathies. Other RET mutations cause MEN 2A syndrome, familial medullary thyroid carcinoma, or Hirschsprung's disease. RET gene expression is also involved in Neuroblastoma. The main diagnosis standards are the acetylcholinesterase study of rectal mucosa and the molecular analysis of RET. In our protocol the rectal biopsy is, therefore, the first approach. RET mutation detection offers the possibility to diagnose MEN 2B predisposition at a pre-clinical stage in familial cases, and to perform an early total prophylactic thyroidectomy. The surgical treatment of MEN 2B is total thyroidectomy with cervical limphadenectomy of the central compartment of the neck. When possible, this intervention should be performed with prophylactic aim before 1 year of age in patients with molecular genetic diagnosis. Recent advances into the mechanisms of RET proto-oncogene signaling and pathways of RET signal transduction in the development of MEN 2 and MTC will allow new treatment possibilities.

Abstract 3101: RET, an estrogen-dependent proto-oncogene, is required for the proliferation of ER-positive breast cancer cells

Abstract Background: RET is a member of the cadherin superfamily, which encodes a receptor tyrosine kinase. RET is a transforming proto-oncogene associated with many endocrine carcinomas. Regulation of RET expression is very complex. The RET promoter can be regulated by many transcription factors, including retinoic acid receptor, Sp1, Nkx2-1, and others. Using a transcriptional profiling assay, our laboratory previously identified RET as an estrogen-induced gene in breast cancer cells. In this study, we investigated the molecular mechanism by which estrogen regulates RET expression in breast cancer, and assessed the function of RET proto-oncogene in breast cancer. Methods: RET expression in breast cancer cell lines was analyzed by Q-RT-PCR, and RET expression in tissue specimens was retrieved from the oncomine database. Transcriptional regulation of RET promoter and distal enhancer elements was analyzed by luciferase assay and chromatin immunoprecipitation (ChIP) after treatment with vehicle or estrogen. Cell proliferation was measured by MTS assay and cell counting. Results: RET was highly expressed in estrogen receptor (ER)-positive breast cancer cell lines and breast cancer tissue specimens compared to ER-negative ones. Expression of RET was induced robustly by estrogen, and this induction was dependent on ER via a primary transcriptional regulatory mechanism, as determined by pretreatment of cycloheximide, ER siRNA, or the pure ER-antagonist, fulvestrant. The proximal promoter of RET gene was not responsive to estrogen treatment and ER did not bind to the proximal promoter. ChIP analysis showed that of nine potential ER binding sites in the RET gene locus, only three bind ER with different efficiency. A 1kb DNA fragment, which is located about 50kb upstream of the transcriptional start site, had an extremely high affinity to ER. When inserted in front of the promoter-luciferase cassette, only this 1kb fragment, but not other eight fragments, enhanced estrogen inducibility and therefore functions as an enhancer acting in cis. This enhancer region has two half estrogen responsive elements (EREs) and a full ERE, which are critical for estrogen-induced RET expression. In a functional assay, siRNA knockdown of RET inhibited estrogen-stimulated cell proliferation in ER-positive breast cancer cells, but not in ER-negative ones. Conclusion: The RET proto-oncogene is induced by estrogen treatment and is required for the proliferation of ER-positive breast cancers. Estrogen-induced RET expression is facilitated by ER binding to a distal enhancer containing novel EREs. This pathway of estrogen regulation of RET gene expression and cell proliferation may be targeted in the future for better treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2011-3101

Reduced RET expression in gut tissue of individuals carrying risk alleles of Hirschsprung's disease

Receptor tyrosine kinase (RET) single nucleotide polymorphisms (SNPs) are associated with the Hirschsprung's disease (HSCR). We investigated whether the amount of RET expressed in the ganglionic gut of human was dependent on the genotype of three regulatory SNPs (-5G>A rs10900296 and -1A>C rs10900297 in the promoter, and C>T rs2435357 in intron 1). We examined the effects of three regulatory SNPs on the RET gene expression in 67 human ganglionic gut tissues using quantitative real-time PCR. Also, 315 Chinese HSCR patients and 325 ethnically matched controls were genotyped for the three SNPs by polymerase chain reaction (PCR) and direct sequencing. The expression of RET mRNA in human gut tissue did indeed correlate with the genotypes of the individuals. The lowest RET expression was found for those individuals homozygous for the three risk alleles (A-C-T/A-C-T), and the highest for those homozygous for the 'wild-type' counterpart (G-A-C/G-A-C), with expression values ranging from 218.32 +/- 125.69 (mean +/- SE) in tissues from individuals carrying G-A-C/G-A-C to 31.42 +/- 8.42 for individuals carrying A-C-T/A-C-T (P = 0.018). As expected, alleles -5A, -1C and intron 1 T were associated with HSCR (P = 5.94 x 10(-31), 3.12 x 10(-24) and 5.94 x 10(-37), respectively) as was the haplotype encompassing the three associated alleles (A-C-T) when compared with the wild-type counterpart G-A-C (chi2 = 155.29, P << 0.0001). To our knowledge, this is the first RET expression genotype-phenotype correlation study conducted on human subjects to indicate common genetic variants in the regulatory region of RET may play a role in mediating susceptibility to HSCR, by conferring a significant reduction of the RET expression.

Effect of silencing RET gene expression on the proliferation of medullary thyroid carcinoma cells

Objective To investigate the effect of silencing RET gene expression on IT (human medullary thyroid carcinoma cell lines) cell proliferation and its mechanism.Methods The double strands DNA containing RET gene was cloned into the pSilencerTM 3,1-H1 neo eukaryotic expression vector,and then was transfected into IT cells expressing high level of RET by lipofactamine.The levels of RET mRNA and protein expression and extracellular signal regulated kinase (ERK) phosphorylation were detected by real-time PCR and Western blotting.TT cell proliferation was tested by cell counting kit-8.Results The recombinant plasmid was successfully constructed as shown by sequencing.After the recombinant plasmid was transfected into TT cells,ret mRNA and RET protein expressions were reduced,along with decreased TT cell proliferation and ERK phosphorylation (P＜0.05).Conclusions The pSilencer-ret plasmid decreases the level of RET expression and inhibits TT cell proliferation,which is involved in ERK pathway.This may provide a new gene therapy approach for medullary thyroid carcinoma. Key words: RNA interfering; Ret gene; Medullary thyroid carcinoma; Gene therapy

Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung’s disease

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung's disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell's procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.