Análisis de mutaciones BRAF y NRAS de tumores tiroideos en pacientes de Argentina / Analysis of BRAF and NRAS mutations in thyroid tumors in patients from Argentina

Resumen Introducción : Se ha descrito que alteraciones molecu lares de las células foliculares tiroideas en el gen BRAF o en NRAS están asociadas con el proceso de carcinogé nesis. Nuestro objetivo fue conocer la frecuencia muta cional de BRAF y NRAS a partir de muestras de punción aspirativa con aguja fina (PAAF) en nuestra población. Métodos : Se analizó por qPCR el estado mutacional de BRAF (codón 600) y NRAS (codón 61) de 193 mues tras obtenidas por PAAF de nódulos sospechosos y se comparó con los datos de la anatomía patológica de 115 pacientes. Resultados : La mutación BRAF se identificó en 40 muestras (74.1%) de las punciones categorizadas como Bethesda VI (n = 54). En las muestras que se correspon dieron con carcinoma papilar de tiroides (CPT) variante clásica por histología (n = 47), el 70% presentó la muta ción, mientras que en los otros subtipos la prevalencia fue más baja (p = 0.013). En muestras de lesión folicular (n = 36), el 50% de los carcinomas foliculares resultaron positivos para NRAS pero solo el 6.7% de los adenomas presentaron esta variación. La presencia de mutación BRAF y CPT se asociaron con metástasis en los gan glios linfáticos (p = 0.014) y mayor riesgo relativo de recurrencia según el Consenso Argentino Intersocietario (RR = 6.77, p = 0.022). No hubo diferencias significativas entre la mutación de BRAF y otras características de agresividad en CPT. Conclusión : La mutación de BRAF y NRAS se observa en un número significativo de CPT y carcinoma folicular, respectivamente, en nuestra población. La mutación BRAF se correlaciona significativamente con metástasis en los ganglios linfáticos.

Abstract Introduction : Molecular alterations in follicular cells in the BRAF or NRAS genes have been reported to be associated with the process of carcinogenesis. Our aim was to determine the mutational frequency of BRAF and NRAS in fine-needle aspiration (FNA) specimens in our population. Methods : The mutational status of BRAF (codon 600) and NRAS (codon 61) was analysed by qPCR in 193 FNA specimens from suspicious nodules and compared with pathological data of 115 patients. Results : BRAF mutation was identified in 40 samples (74.1%) of FNAs classified as Bethesda VI (n = 54). In samples histologically diagnosed as classic papillary thyroid carcinoma (cPTC, n = 47), mutation was observed in 70% of cases, while in other subtypes the prevalence was lower (p = 0.013). In FNA specimens of follicular lesions (n = 36), positivity for NRAS was found in 50% of the follicular carcinomas (FTCs), but only in 6.7% of adenomas. Finally, there was a significant correlation between BRAF and PTC with lymph-node metastasis (p = 0.014) and increased relative risk of recurrence based on the Argentine Intersociety Consensus (RR = 6.77, p = 0.022). No significant differences were found between BRAF mutation and other features of aggressiveness in PTC. Conclusion : BRAF and NRAS mutations are observed in a significant number of PTCs and FTCs, in our popu lation. There is a significant correlation between BRAF mutation and lymph-node metastasis.

Correlation of gene mutations and microsatellite instability in colorectal cancer tissues with clinicopathological features of patients / 肿瘤研究与临床

Objective:To explore KRAS, NRAS, BRAF gene mutations and microsatellite instability(MSI) in colorectal cancer tissues as well as their correlation with the clinicopathological characteristics of patients.Methods:The clinicopathological data of 473 colorectal cancer patients in Shanxi Province Cancer Hospital from October 2020 to May 2021 were retrospectively analyzed. The mutation status of KRAS, NRAS and BRAF gene in the paraffin tissues were detected by using amplification refractory mutation system (ARMS) method. Polymerase chain reaction (PCR)-capillary electrophoresis was used to analyze MSI status, and the correlation of the clinicopathological characteristics of patients with gene mutations and MSI status was analyzed.Results:The mutation rates of KRAS, NRAS and BRAF were 45.03% (213/473), 2.96% (14/473) and 5.50% (26/473), respectively in 473 patients with colorectal cancer. No case harbored both 2 gene mutations was detected. The mutation rate of KRAS gene in well differentiated adenocarcinoma was higher than that in poorly differentiated adenocarcinoma [47.4% (175/369) vs. 36.5% (38/104), χ2 = 3.89, P = 0.049]. NRAS mutation rate in female was higher than that in male [5.0% (10/202) vs. 1.5% (4/271), χ2 = 4.86, P = 0.027], and the NRAS mutation rate in patients with tumor diameter ≤ 3 cm was higher than that in those with tumor diameter >3 cm [7.1% (7/98) vs. 1.9% (7/375), P = 0.013]. BRAF mutation rate of tumors located in colon was higher than that in rectum [11.7% (20/171) vs.2.0% (6/302), χ2 = 19.81, P < 0.001]; BRAF mutation rate in poorly differentiated tumor was higher than that in well differentiated tumor [10.6% (11/104) vs. 4.1% (15/369), χ2 = 6.62, P = 0.010]; BRAF mutation rate in patients with mucus was higher than that in those without mucus [10.9% (11/101) vs. 4.0% (15/372), χ2 = 7.19, P = 0.007]; BRAF mutation rate in patients with lymphatic metastasis was higher than that in patients without lymphatic metastasis [8.2% (15/182) vs.3.8% (11/291), χ2 = 4.29, P = 0.038]. The incidence of high frequency MSI (MSI-H) in 473 colorectal cancer tissues was 7.19% (34/473). The incidence of MSI-H in colon was higher than that in rectum [14.0% (24/171) vs. 3.3% (10/302), χ2 = 18.82, P < 0.001]; the incidence of MSI-H in patient with poor differentiated tumor was higher than that in those with well differentiated tumor [17.3% (18/104) vs. 4.3% (16/369), χ2 = 20.46, P < 0.001]; the incidence of MSI-H in patients with mucus was higher than that in those without mucus [11.9% (12/101) vs. 5.9% (22/372), χ2 = 4.24, P = 0.039]; and the incidence of MSI-H in patients without lymphatic metastasis was higher than that in patients with lymphatic metastasis [10.0% (29/291) vs. 2.7% (5/182), χ2 = 8.75, P = 0.003]. In addition, the incidence of MSI-H was on the rise in patients with BRAF mutation ( P < 0.001). Conclusions:KRAS, NRAS, BRAF gene mutations and MSI status are correlated with the clinicopathological characteristics of patients with colorectal cancer; there is a close relationship between MSI-H and BRAF mutation.

Extended?spectrum of KRAS and NRAS mutations in lung cancer tissue specimens obtained with bronchoscopy

Background: Mutations in the RAS genes, HRAS, KRAS, and NRAS, are the most common modifications in many types of human tumors and are found in approximately 30% of all human cancers. These mutations are usually found in codons 12, 13, or 61. Methods: The aim of this study is to evaluate mutations in codons 59, 117, and 146 of KRAS and NRAS genes in addition to codons 12,13, and 61 of KRAS gene in lung cancer tissue specimens obtained with bronchoscopy. KRAS and NRAS mutation analyses with pyrosequencing were performed on DNA isolated from formalin?fixed paraffin?embedded (FFPE) tissue samples of 64 patients histopathologically diagnosed as lung cancer after bronchoscopic biopsy. Results: In all, 20 patients (31.2%) had mutations in KRAS gene (8/27 squamous cell carcinoma, 8/11 adenocarcinoma, 3/16 small cell carcinoma, and 1/1 pleomorphic carcinoma). The most common mutation in codon 12 was in c.35G>T (G12V). When the mutation rate of adenocarcinoma (72.7%) and squamous cell carcinoma (22.9%) patients was compared with each other, a statistically significant difference was observed (P = 0.008). There were no mutations in codons 59, 117, or 146 of KRAS and NRAS genes in patients with lung cancer. Conclusion: In this study, we firstly examined mutations in codons 59, 117, and 146 of KRAS and NRAS genes in addition to codons 12, 13, and 61 of KRAS gene in Turkish lung cancer patients both in non?small cell lung cancer and small cell lung cancer. Although no mutation was detected in codons 59, 117, and 146 of KRAS and NRAS genes, the frequency of KRAS gene mutation was higher than the rate of mutation in both Asian and Western countries, and multicenter studies including more cases should be performed to further explore our results.

Analysis of Coexisting Gene with NRAS in Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients.@*METHODS@#High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated.@*RESULTS@#A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons.@*CONCLUSION@#The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.

microRNA-181a-5p impedes the proliferation, migration, and invasion of retinoblastoma cells by targeting the NRAS proto-oncogene

Abstract Objectives Accumulating research have reported that microRNAs (miRNAs) play important roles in Retinoblastoma (RB). Nonetheless, the function and underlying mechanism of miR-181a-5p in RB remain ambiguous. Methods The relative expression levels of miR-181a-5p and NRAS mRNA were detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). RB cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) and 5′-Bromo-2′-deoxyuridine (BrdU) assays. Transwell assays and flow cytometry were performed to detect the migration, invasion, and apoptosis of RB cells. The interaction between miR-181a-5p and NRAS was explored using luciferase experiments, western blotting, and qRT-PCR. Results miR-181a-5p expression was found to be decreased in RB tissues and cell lines, and its expression was correlated with unfavorable pathological features of the patients. In vitro experiments revealed that miR-181a-5p reduced RB cell proliferation, migration, and invasion while enhancing apoptosis. Further research confirmed that NRAS is a direct target of miR-181a-5p. miR-181a-5p inhibited NRAS expression at both the mRNA and protein levels. Co-transfection of pcDNA-NRAS or NRAS small interfering RNA (siRNA) reversed the effects of miR-181a-5p mimics or miR-181a-5p inhibitors on RB cells. Conclusion miR-181a-5p was significantly downregulated during the development of RB, and it suppressed the malignant behaviors of RB cells by targeting NRAS.

GIANT CONGENITAL MELANOCYTIC NEVI – A RARE CASE REPORT

Giant congenital melanocytic nevi (GCMN) are the melanocytic lesions that arise due to the abnormal migration of melanoblasts during the embryogenesis, affecting approximately one in 500,000 live births. There is gain-of-function mutation in the NRAS gene is usually associated with GCMN, causing abnormal proliferation of embryonic melanoblasts that are usually present since birth with change in their morphological characteristics with time, and increasing in their size reaching upto a diameter ? 20 cm in adulthood. These are characterized by dark brown to black in color with irregular margins and having verrucous surfaces, with or without satellite lesions which are present beyond the periphery of the central lesion. Depending on their distribution these can be classified as bathing trunk, coat-sleeve, or stocking nevi. We hereby report a case of a sixteen year old female presenting with GCMN. The development of a malignant neoplasm on same lesion is only absolute indication for surgery in GCMN

A Retrospective Study Of Kras And Nras Mutations In Metastatic Colo-Rectal Cancer Patients And Treatment Outcomes With Cetuximab- Or Bevacizumab-Containing Regimens In Central India

Background: The presence of a Kristen-Rat Sarcoma (KRAS) and NRAS activating mutations in metastatic CRC (mCRC) were significantly associated with absence of response to agents targeting epidermal growth factor receptor(EGFR). Our study aims to describe the pattern of KRAS and NRAS mutations and effectiveness of Cetuximab- and Bevacizumab-containing regimens in mCRC patients. Methodology: In this retrospective observational study, a total of 156 mCRC patients data were evaluated, out of which 124 patient’s KRAS and NRAS mutation status was known. We evaluated demographic and clinical characteristics and progression-free survial(PFS) of mCRC patients treated with cetuximab-or bevacizumab-containing regimens. Results: Median age was 56 yrs(18-78yrs). Total 124 patient’s mutation status was known,out of which 34 (27%) were positive for KRAS and 4 (3%) for NRAS. Overall, there were 83 (67%) males and 41 (33%) females. In KRAS positive group, 26 (76%) were males and 08 (24%) females. Bevacizumab treated patients(69.2%) mostly received oxaliplatin-based backbone, whereas, cetuximab treated pateints(56.8%) mostly received irinotecan-based chemotherapy. There was no significant difference in PFS between cetuximab-and bevacizumab-based treated patients. Conclusion: Efforts to obtain tissue samples should be encouraged for KRAS and NRAS mutation testing in mCRC patients to provide a molecular basis to treat with available anti-EGFR monoclonal antibodies in mutation wild-type patients.

Genetic Alterations among Korean Melanoma Patients Showing Tumor Heterogeneity: A Comparison between Primary Tumors and Corresponding Metastatic Lesions / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Melanoma is a highly heterogeneous neoplasm, composed of subpopulations of tumor cells with distinct molecular and biological phenotypes and genotypes. In this study, to determine the genetic heterogeneity between primary and metastatic melanoma in Korean melanoma patients, we evaluated several well-known genetic alterations of melanoma. In addition, to elucidate the clinical relevance of each genetic alteration and heterogeneity between primary and metastatic lesions, clinical features and patient outcome were collected. MATERIALS AND METHODS: In addition to clinical data, BRAF, NRAS, GNAQ/11 mutation and KIT amplification data was acquired from an archived primary Korean melanoma cohort (KMC) of 188 patients. Among these patients, 43 patients were included for investigation of tumor heterogeneity between primary melanoma and its corresponding metastatic lesions. RESULTS: Overall incidence of genetic aberrations of the primary melanomas in KMC was 17.6% of BRAF V600, 12.6% of NRAS mutation, and 28.6% of KIT amplification. GNAQ/11 mutation was seen in 66.6% of the uveal melanoma patients. Patients with BRAF mutation were associated with advanced stage and correlated to poor prognosis (p < 0.01). Among 43 patients, 55.8% showed heterogeneity between primary and metastatic lesion. The frequency of BRAF mutation and KIT amplification significantly increased in the metastatic lesions compared to primary melanomas. GNAQ/11 mutation showed 100% homogeneity in uveal melanoma patients. CONCLUSION: Our data demonstrated heterogeneity between primary melanomas and corresponding metastatic lesions for BRAF, NRAS mutation and KIT amplification. However, GNAQ/11 mutation was genetically homogeneous between primary and metastatic melanoma lesions in uveal melanoma.

Retrospective analysis of KRAS, NRAS and BRAF gene mutations and clinicopathological features in patients with colorectal cancer / 上海交通大学学报（医学版）

Objective·To study the correlation between KRAS,NRAS and BRAF mutations and the clinicopathological features in patients with coloreetal cancer (CRC).Methods·The 461 paraffin-embedded CRC tissues were collected.The mutations in hotspot region of KRAS,NRAS and BRAF genes were investigated using amplification refractory mutation system.The relationship between the mutation rates of each gene and the clinicopathological characteristics in CRC patients were analyzed.TheKRAS and BRAF mutation profile and the impact on promoter methylation of the downstream genes were further investigated in both CRC tissues and cell lines through literatures and the American Type Culture Collection.Results·KRAS,NRAS and BRAF mutation rates in CRC tissues were 44.0％,6.1％ and 5.2％,respectively.KRAS mutations in the right colon were remarkably higher than the left colon (P=0.000).NRAS mutations were more likely to occur in male patients than female patients (P=0.002).BRAF mutation was closely correlated with age,tumor differentiation,tumor location and nerve invasion,but was exclusive of 203 KRAS mutated samples.Contusion·KRAS,NRAS and BRAF mutations were significantly correlated with the clinicopathological features of CRC,and the mutation incompatibility was observed between KRAS and BRAF genes.

Retrospective analysis of KRAS, NRAS and BRAF gene mutations and clinicopathological features in patients with colorectal cancer / 上海交通大学学报（医学版）

Objective: To study the correlation between KRAS, NRAS and BRAF mutations and the clinicopathological features in patients with colorectal cancer (CRC). Methods: The 461 paraffin-embedded CRC tissues were collected. The mutations in hotspot region of KRAS, NRAS and BRAF genes were investigated using amplification refractory mutation system. The relationship between the mutation rates of each gene and the clinicopathological characteristics in CRC patients were analyzed. The KRAS and BRAF mutation profile and the impact on promoter methylation of the downstream genes were further investigated in both CRC tissues and cell lines through literatures and the American Type Culture Collection. Results: KRAS, NRAS and BRAF mutation rates in CRC tissues were 44.0%, 6.1% and 5.2%, respectively. KRAS mutations in the right colon were remarkably higher than the left colon (P=0.000). NRAS mutations were more likely to occur in male patients than female patients (P=0.002). BRAF mutation was closely correlated with age, tumor differentiation, tumor location and nerve invasion, but was exclusive of 203 KRAS mutated samples. Conclusion: KRAS, NRAS and BRAF mutations were significantly correlated with the clinicopathological features of CRC, and the mutation incompatibility was observed between KRAS and BRAF genes.

Analysis of NRAS gene mutations and prognostic factors in patients with acral melanoma / 中华皮肤科杂志

Objective To detect NRAS gene mutations in patients with acral melanoma, and to analyze their relationship with the prognosis of acral melanoma. Methods Clinical and pathological data were collected from 55 patients with pathologically diagnosed acral melanoma. DNA was extracted from paraffin?embedded specimens from lesions of the 55 patients and 15 patients with nevus. PCR and direct DNA sequencing were performed to detect NRAS gene mutations. Univariate and multivariate analyses were performed using the Cox′s proportional hazards regression model. Results Of the 55 patients, 6(10.9%)carried the Q61R mutation in codon 61 of the NRAS gene. No mutations were found in exon 1 or 2 of the NRAS gene in any of these paraffin?embedded specimens, and none of the pigmented nevus specimens harbored NRAS gene mutations. Of the 6 patients carrying NRAS gene mutations, 4 had lymph node metastasis. Multivariate Cox regression analysis showed that independent factors of poor prognosis included advanced clinical stage(RR = 2.54, 95% CI: 1.062- 6.066, P < 0.05), not receiving surgical resection(RR = 2.98, 95% CI:1.316- 3.525, P < 0.05), and carrying NRAS gene mutations (RR = 2.73, 95% CI: 0.932- 3.257, P < 0.05). Conclusions NRAS gene mutations may be associated with lymph node metastasis in patients with acral melanoma. The prognosis of acral melanoma may be associated with clinical staging, treatment strategies and NRAS gene mutations. Additionally, NRAS gene mutations may serve as a new index for predicting prognosis of acral melanoma.

Effects of miR-145 on the proliferation, apoptosis and cell cycle of a human keratinocyte cell line HaCaT / 中华皮肤科杂志

Objective To investigate the regulatory effects of miR-145 on the proliferation,cell cycle and apoptosis of a human keratinocyte cell line HaCaT.Methods miR-145 mimics and negative control (NC) mimics were chemically synthesized and then transiently transfected into HaCaT cells respectively.After additional culture for different durations,real-time PCR was performed to determine the expression level of miR-145,MTS assay to estimate cell proliferation,and flow cytometry to detect cell apoptosis and cycle.Luciferase assay,real-time PCR and Western blot were conducted to determine whether NRAS was the target gene of miR-145.Results The miR-145 expression level in miR-145 mimic-transfected cells increased by 85.00 ± 1.21 folds compared with NC mimic-transfected cells (t =115.90,P ＜ 0.0001).The transfection with miR-145 mimics significantly inhibited the proliferation of HaCaT cells (F =8.76,P =0.008),and the inhibitory effect significantly varied with the duration (24-96 hours) of culture after transfection,with no interaction effect between the transfection with miR-145 mimics and culture duation (F =1.21,P =0.18).Compared with NC mimic-transfected cells,those transfected with miR-145 mimics showed a significant increase in the proportion of early apoptotic cells (18.9％ ± 4.1％ vs.4.3％ ± 1.2％,t =7.126,P ＜ 0.01),late apoptotic cells (9.3％ ± 2.3％ vs.3.6％ ± 1.6％,t =12.38,P ＜ 0.01),G1-phase cells (85.83％ ± 5.2％ vs.62.08％ ± 6.23％,t =11.78,P =0.007),but a significant decrease in the percentage of G2-phase cells (6.26％ ± 1.2％ vs.19.36％ ± 3.45％,t =7.610,P =0.017) and S-phase cells (7.91％ ± 1.3％ vs.18.56％ ± 5.23％,t =7.230,P=0.019).As luciferase assay showed,luciferase activity was significantly lower in HaCaT cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-wild carrying the wild-type 3'UTR of NRAS than in those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-wild (t =11.09,P =0.008),but similar between cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-mut carrying the mutant-type 3'UTR of NRAS and those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-mut (P ＞ 0.05).Real-time PCR and Western blot revealed that the overexpression of miR-145 mimics had no significant effect on NRAS mRNA expression (P ＞ 0.05),but significantly inhibited NRAS protein expression (1.52 ± 0.07 vs.0.20 ± 0.02,t =28.43,P＜ 0.01).Conclusion miR-145 might inhibit proliferation and promote apoptosis of HaCaT cells by influencing cell cycle via NRAS.

CCL18 downregulates the expression of miR98 in breast cancer cells via N-Ras/c-myc/lin28 pathway / 西安交通大学学报(医学版)

Objective To explore whether CCL18 is involved in regulating the expression of miRNAs in breast cancer.Methods The expression profile of miRNAs in the breast cancer cell following CCL18 treatment was determined by miRNAs microarray analysis.Then we performed QRT-PCR and Luciferase Reporter Assay to validate the results from the miRNAs microassay.We used transient transfection to change the expression of miR98 and c-myc in breast cancer cells.We then used QRT-PCR and Western blot to analyze the mechanism by which CCL18 downregulates the expression of miR98 in breast cancer cells.Results miRNAs microarray analysis showed that cells treated with CCL18 differentially expressed 20 miRNAs genes compared with those in the control group. Our QRT-PCR and Luciferase Reporter Assay confirmed the result.The mRNA and protein expressions of C-myc and lin28 were increased after CCL18 stimulation in breast cancer cells.Transfection with c-myc siRNAs rescued the increase of lin28 and loss of miR98 expression caused by CCL18 stimulation.Our results also showed that CCL18 could upregulate the expression of N-Ras at post-transcription level.Conclusion CCL18 downregulates the expression of miR98 via N-Ras/c-myc/lin28 pathway.The downregulated miR98 increases the expression of N-Ras after transfection,which further activates c-myc/lin28 pathway and forms a positive feedback loop.

NRAS Mutations in de novo acute leukemia: Prevalence and clinical significance.

The activating mutations of the Ras gene or other abnormalities in Ras signaling pathway lead to uncontrolled growth factor-independent proliferation of hematopoietic progenitors. Oncogenic mutations in NRAS gene have been observed with variable prevalence in hematopoietic malignancies. In the present study, NRAS mutations were detected using bidirectional sequencing in 264 acute leukemia cases — 129 acute lymphocytic leukemia (ALL) and 135 acute myeloid leukemia (AML) and 245 age- and gender-matched controls. Missense mutation was observed only in the 12th codon of NRAS gene in 4.7% of AML and 3.16% of ALL cases. The presence of NRAS mutation did not significantly influence blast % and lactate dehydrogenase (LDH) levels in AML patients. When the data were analyzed with respect to clinical variables, the total leukocyte count was elevated for mutation positive group, compared to negative group. In AML patients with NRAS mutations, 60% failed to achieve complete remission (CR), as compared to 34.8% in mutation negative group. These results indicated that NRAS mutations might confer poor drug response. In AML, disease free survival (DFS) in NRAS mutation positive group was lesser, compared to mutation negative group (9.5 months vs. 11.68 months). In ALL patients, DFS of NRAS mutation positive group was lesser than mutation negative group (9.2 months vs. 27.5 months). The CR rate was also lower for mutation-positive patients group, compared to mutation-negative group. In conclusion, these results suggested that presence of NRAS mutation at 12th codon was associated with poor response and poorer DFS in both ALL and AML.

Prevalence of BRAF and NRAS Mutations and a Comparative Analysis in Primary and Metastatic Melanoma of Korean Patients / 이화의대지

OBJECTIVES: The aim of this study is to verify the status and the clinical significance of BRAF and NRAS mutations in patients of one of the university hospitals in Korea. METHODS: Polymerase chain reaction (PCR) amplification and direct sequencing were performed for the analysis of melanoma samples (n=22) for the detection of mutations in exon 15 of the BRAF gene, and exons 2 and 3 of the NRAS gene in genomic DNA. Mutations of the BRAF gene were correlated with the clinicopathologic features of patients and the BRAF mutation status was compared in 18 paired primary and metastatic tumors. RESULTS: Incidence of somatic mutations within the BRAF and NRAS genes was 27.3% (6/22) and 0% (0/22), respectively. Age, gender, Breslow thickness, and ulceration did not show correlation with BRAF mutations. Among 18 patients with metastasis, BRAF mutation was detected in 22.2% of cases (4/18), and all four cases with BRAF mutations were identified in metastatic lymph node tissues. BRAF mutations were only found in lymph node metastases, which was statistically significant (28.6% vs 0%, P<0.01). CONCLUSION: The incidence of BRAF mutation is as low as in other Asian reports and the NRAS mutation was not found in patients of our institute.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

Effect of N-Ras by RNA interference on radiosensitivity of hepatoma carcinoma cell MHCC97-H line / 中华放射医学与防护杂志

Objective To investigate the radiosensitivity of silencing N-Ras by RNA interference in hepatoma carcinoma cell MHCC-97.Methods N-Ras RNA interference (RNAi) vector was constructed by using pcDNA 6.2-GW/EmGFP-mir plamid.The RNAi effect was detected by RT-PCR,Western bolt,immunohistochemisty and MTT method.Survival curve for each cell line were obtained by measuring the clone forming abilities of irradiated cell populations.Results After silencing the N-Ras by RNAi,The expression level of N-Ras mRNA,N-Ras protein,immunohistochemisty were decreased 96.9% ±0.159%(t=40.377,P＜0.05),89.8%±0.012% (t=31.595,P＜0.05),90%,respectively,and The survival of hepatoma carcinoma cell MHCC-97 line were inhibited 21.9% (F = 4.63,P < 0.05).Which have significant difference in statistics.The SER of hepatoma carcinoma cell MHCC-97 line after interference was 1.15.Conclusions RNAi targeting silence N-Ras may increase the radiosensitivity of hepatoma carcinoma cell MHCC-97 line.

Mutation of the N-ras Gene in a Patient Suffering from the Blast Phase of Chronic Myelogenous Leukemia / 대한혈액학회지

The blast phase in chronic myelogenous leukemia (CML) is associated with mutation of several genes. It is well known that p53 gene mutation plays a key role in the myeloid or lymphoid blast phase of CML. But for the case of the N-ras gene, the association between N-ras mutations and the blast phase of CML is not yet known. We report here on a case of detecting N-ras point mutation without p53 mutation in a 64 year-old man who suffered from the lymphoblastic blast phase of CML.

Fatal outcome of a young woman with papillary thyroid carcinoma and graves' disease: possible implication of "cross-signalling" mechanism / Desfecho Fatal de uma Paciente com Carcinoma Papilífero de Tiróide e Doença de Graves: possível Implicação do Mecanismo de Sinalização Cruzada

A 29 yrs-old patient was referred to our hospital due to generalized convulsions. She had hyperthyroidism treated with methimazole. Her MRI showed 4 metastatic lesions in the brain. She had a goiter with a "cold" nodule and a palpable ipsilateral lymph node. The FNAB disclosed a papillary thyroid carcinoma. Under 5 mg of MMI treatment, she had a subclinical hyperthyroidism and TRAb were 47.8 percent (n.v. < 10 percent). The CT scan also showed lung metastasis. She underwent a total thyroidectomy with a modified neck dissection and she received an accumulated radioiodine dose of 700 mCi during the following two years. She died from the consequences of multiple metastatic lesions. Studies were performed in DNA extracted from paraffin-embedded tissue from the tumor, the metastatic lymph node and the non-tumoral thyroid. The genetic analysis of tumoral DNA revealed point mutations in two different genes: the wild type CAA at codon 61 of N-RAS mutated to CAT, replacing glycine by histidine (G61H) and the normal GCC sequence at codon 623 of the TSHR gene was replaced by TCC, changing the alanine by serine (A623S). In the non-tumoral tissue no mutations were found. In vitro studies showed a constitutive activation of the TSHR. It is very probable that this activating mutation of the TSHR is unable to reach the end point of the PKA cascade in the tumoral tissue. One possibility that could explain this is the presence of a cross-signaling mechanism generating a deviation of the TSH receptor cascade to the more proliferative one involving the MAPKinase, giving perhaps a more aggressive behavior of this papillary thyroid cancer.

Paciente de 29 anos foi encaminhada ao Hospital de Clínicas por causa de convulsões generalizadas. Apresentava hipertiroidismo tratado com metimazol (MMI). A ressonância magnética mostrava quatro lesões metastáticas cerebrais. Possuía bócio com nódulo frio e linfonodo palpável ipsilateral. Usando 5 mg de MMI, a paciente apresentava hipertiroidismo subclínico e TRAb = 47,8 por cento (normal < 10 por cento). A tomografia computadorizada também mostrava metástases pulmonares. A paciente foi submetida a tiroidectomia total com dissecção cervical modificada e recebeu dose acumulada de radioiodo de 700 mCi durante o período de dois anos. Foi analisado o DNA extraído de tecido emblocado em parafina do tumor, do linfonodo metastático e de tecido tiroidiano não-tumoral. Foram encontradas mutações pontuais em dois genes: uma substituição do genótipo selvagem CAA no códon 61 de /N-RAS/ por CAT, substituindo a glicina pela histidina (G61H) e uma substituição da seqüência normal GCC no códon 623 do gene TSHR por TCC, trocando a alanina pela serina (A623S). Não foram encontradas mutações no tecido não-tumoral. Estudos in vitro mostraram ativação constitutiva de TSHR. Já que esta mutação ativadora de TSHR foi incapaz de atingir o final da cascata PKA no tecido tumoral, sugere-se que um mecanismo de cross-signaling possa explicar o desvio da cascata do receptor de TSH para outra mais proliferativa, envolvendo MAPKinase e levando ao comportamento mais agressivo deste câncer papilífero.