Correlation of KRAS gene mutation and programmed death receptor ligand 1 expression with prognosis of first-line concurrent chemoradiotherapy in patients with locally advanced non-small cell lung cancer / 肿瘤研究与临床

Objective:To investigate the relationship between KRAS gene mutation, programmed death receptor ligand 1 (PD-L1) expression and prognosis of first-line concurrent chemoradiotherapy in patients with locally advanced non-small cell lung cancer.Methods:The clinical data of 50 patients with locally advanced non-small cell lung cancer who were admitted to Nanping First Hospital from January 2018 to December 2021 were retrospectively analyzed. All patients were treated with first-line concurrent chemoradiotherapy. Tissue samples of patients were obtained and paraffin embedded before treatment. Real-time fluorescence quantitative polymerase chain reaction was used to detect the type of KRAS gene mutation in tissues before treatment, and the expression of PD-L1 was determined by immunohistochemistry (the percentage of positive cells in tumor cells ≥1% was positive), and the relationship between KRAS gene status, PD-L1 expression and clinical characteristics and short-term efficacy of patients was analyzed. Patients were followed up for 1 year, and progression-free survival (PFS) curves were plotted by Kaplan-Meier method, and log-rank test was used for comparison. Univariate and multivariate Cox proportional hazards models were used to analyze the influencing factors of PFS.Results:Among the 50 patients, 11 (22.00%) were KRAS mutant, and 36 (72.00%) were PD-L1 positive. Among the 11 patients with KRAS mutation, there were 2 cases of codon 13 mutation and 9 cases of codon 12 mutation in exon 2. The objective response rate (ORR) and clinical control rate (DCR) were 76.00% (38/50) and 86.00% (43/50). There were no significant differences in patients' age, pathological type, TNM stage, ORR and DCR between KRAS mutant group and KRAS wild type group (all P > 0.05). The proportions of male patients [72.73% (8/11) vs. 38.46% (15/39)], patients with smoking history [90.91% (10/11) vs. 20.51% (8/39)] and patients with PD-L1 positive expression [100.00% (11/11) vs. 64.10% (25/39)] in KRAS mutant group were higher than those in KRAS wild type group (all P < 0.05). There were no significant differences in patients' age, pathological type, gender, smoking history, TNM stage, ORR and DCR between PD-L1 positive group and PD-L1 negative group (all P > 0.05). The median PFS time of patients in KRAS mutant group and wild type group was 8.75 and 11.32 months, and the difference in PFS between the two groups was statistically significant ( P = 0.039). The median PFS time of patients with PD-L1 positive and negative was 10.19 and 11.16 months, and there was no statistical significance in PFS between the two ( P = 0.116). Multivariate Cox regression analysis showed that KRAS gene mutation was an independent risk factor for PFS in patients with locally advanced NSCLC after first-line concurrent chemoradiotherapy ( HR = 1.449, 95% CI 1.071-1.196, P = 0.017). PD-L1 expression, smoking history and gender were not independent influencing factors for PFS (all P > 0.05). Conclusions:KRAS gene status is closely related to the prognosis of patients with locally advanced non-small cell lung cancer treated with first-line concurrent chemoradiotherapy, while PD-L1 expression is not.

Value of 18F-FDG PET/CT radiomics for predicting KRAS gene mutations in non-small cell lung cancer / 中华核医学与分子影像杂志

Objective:To assess the predictive efficacy of 18F-FDG PET/CT-based radiomics models for the mutation status of Kirsten rats sarcoma viral oncogene homolog (KRAS) in patients with non-small cell lung cancer (NSCLC). Methods:From January 2016 to January 2021, the 18F-FDG PET/CT images and KRAS testing of 258 NSCLC patients (180 males, 78 females; age: 33-91 years) in the First Affiliated Hospital of the Air Force Military Medical University were retrospectively analyzed. Patients were randomly divided into training set ( n=180) and validation set ( n=78) in the ratio of 7∶3. Tumor lesions on PET and CT images were drawn respectively, and the radiomics features of PET and CT lesions were extracted. The radiomics features were screened by least absolute shrinkage and selection operator (LASSO). CT radiomics score (RS) model, PET/CT RS model and composite models of PET/CT RS combined with screened clinical information were eventually developed. ROC curves were used to assess the predictive efficacy of these models. Results:The CT RS model included 4 radiomics features and the PET/CT RS model included 4 CT radiomics features and 8 PET radiomics features. The CT RS model and the PET/CT RS model both had significant differences in RS between KRAS mutant and wild-type patients in the training set and validation set ( z values: from -8.30 to -4.10, all P<0.001). In predicting KRAS mutations, the composite model of PET/CT RS combined with age showed AUCs of 0.879 and 0.852 in the training and validation sets respectively, which were higher than those of the CT RS model (0.813 and 0.770) and the PET/CT RS model (0.858 and 0.834). The accuracy of the composite model of PET/CT RS combined with age were 81.67%(147/180) and 79.49%(62/78) in the training set and validation set respectively, which were also higher than those of the CT RS model (75.00%(135/180) and 74.36%(58/78)) and the PET/CT RS model (78.89%(142/180) and 78.21%(61/78)). Conclusion:Models based on radiomics features can predict KRAS gene mutation status, and the composite model combining PET/CT RS and age can improve the prediction performance.

BRAF and NRAS prognostic values in conjunctival melanoma: analysis and literature review / Análise do valor prognóstico dos marcadores BRAF e NRAS no melanoma da conjuntiva e revisão da literatura

ABSTRACT Purpose: Conjunctival melanoma is a rare and aggressive tumor with a propensity for regional and distant metastases. This study aimed to analyze BRAF/NRAS markers in conjunctival melanoma and their relationship with tumor recurrences and patient prognosis. Methods: This retrospective, observational, single-center study included consecutive patients with an anatomopathological diagnosis of conjunctival melanoma, registered between January 1992 and December 2019. BRAF/NRAS mutations were analyzed using cobas®4800 kit (Roche®) in samples obtained by excisional or map biopsy. Additionally, the presence of other associated precancerous or tumor lesions was assessed. Results: A total of 12 patients with positive histological samples for conjunctival melanoma were included (7 women, 5 men), with a mean age at diagnosis of 60 years and a mean evolution time of 6.38 ± 3.4 years. BRAF V600E mutation was observed in three biopsies (25%), similar to NRAS Q61X (25%). Recurrences occurred in all patients with positive BRAF or NRAS mutation, and five of these patients developed systemic dissemination (83.33%). Moreover, four of six patients with mutated BRAF or NRAS (66.66%) had histopathological findings of tumor or precancerous lesions. Conclusions: BRAF and NRAS mutations may be risk factors for recurrence and shorter survival in conjunctival melanoma, which would make these patients candidates for targeted therapies and comprehensive and individualized follow-up. All these data warrant standardized prospective studies.

RESUMO Objetivo: O melanoma da conjuntiva é um tumor raro e agressivo, com propensão à disseminação metastática regional e distante. Este estudo tem como objetivo analisar os marcadores BRAF e NRAS no melanoma da conjuntiva e sua relação com recidivas tumorais e com o prognóstico do paciente. Métodos: Este foi um estudo retrospectivo, observacional e unicêntrico de pacientes consecutivos com diagnóstico anatomopatológico de melanoma da conjuntiva feito entre janeiro de 1992 e dezembro de 2019. As mutações BRAF e NRAS foram analisadas com o kit cobas® 4800 (Roche®) em amostras obtidas através de biópsia excisional ou por mapa. Além disso, foi avaliada a presença de lesões pré-cancerosas ou tumorais associadas. Resultados: Foram incluídos 12 pacientes com amostras histológicas positivas para melanoma da conjuntiva (7 mulheres e 5 homens), com idade média ao diagnóstico de 60 anos e tempo médio de evolução de 6,38 ± 3,4 anos. A mutação BRAF V600E foi encontrada em 3 biópsias (25%), bem como a NRAS Q61X (25%). Ocorreram recidivas em todos os pacientes positivos para mutações de BRAF ou NRAS e 5 desses pacientes desenvolveram disseminação sistêmica (83,33%). Além disso, 4 dos 6 pacientes com BRAF ou NRAS mutante (66,66%) apresentaram achados histopatológicos de lesões tumorais ou pré-cancerosas. Conclusões: As mutações BRAF e NRAS podem ser fatores de risco para recorrência e menor sobrevida no melanoma da conjuntiva, o que tornaria esses pacientes candidatos a terapias direcionadas e a um acompanhamento mais abrangente e individualizado. Todos esses dados justificam mais estudos prospectivos padronizados.

Clinical value of circulating tumor cell typing and RAS/RAF gene detection in colorectal cancer / 中国医师杂志

Objective:To investigate the correlation between circulating tumor cell (CTC) detection, RAS/RAF gene mutation and clinicopathological characteristics in patients with colorectal cancer (CRC).Methods:The Amplification Refractory Mutation System (ARMS)-polymerase chain reaction (PCR) were used to detect the gene mutation in the tumor tissues of 138 CRC patients in the Third Affiliated Hospital of Sun Yat-sen University from May 2017 to May 2020. At the same time, the venous blood of 138 patients was collected and enriched for CTC genotyping by mRNA in situ hybridization. The correlation between CTC, RAS/RAF gene mutation and clinicopathological features of CRC patients was analyzed.Results:The mutation rates of KRAS, NRAS and BRAF genes were 48.6%(67/138), 5.1%(7/138) and 1.4%(2/138), respectively; The overall positive rate of CTC was 84.1%(116/138). The positive rates of different CTC types were: 23.1%(32/138) in epithelial type, 71.7%(99/138) in mixed type and 12.3%(17/138) in interstitial type respectively. The positive rate of CTC in CRC patients with clinical stage Ⅲ-Ⅳ, lymph node metastasis (N1-N3) and distant metastasis (M1) was significantly higher than that in CRC patients with stage Ⅰ-Ⅱ, no lymph node metastasis (N0) and no distant metastasis (M0) (all P<0.05). The total number of CTC, mixed CTC and interstitial CTC were positively correlated with clinical stage, lymph node metastasis and distant metastasis (all P<0.05). RAS/RAF gene mutation, gender, age, tumor location and tumor differentiation did not affect the positive rate of CTC (all P>0.05). Conclusions:The results of CTC typing are of great research significance for comprehensive treatment, prognosis assessment and stratified management of CRC, among which the interstitial type of CTC may be a high risk factor for the recurrence and metastasis of CRC.

Correlation between RAS and BRAF V600E gene mutations and clinicopathological characteristics of colorectal cancer / 肿瘤研究与临床

Objective:To investigate the correlation between KRAS, NRAS and BRAF V600E gene mutations and the clinicopathological characteristics of patients with colorectal cancer.Methods:Specimens from 217 patients with colorectal cancer who underwent surgical resection and were pathologically confirmed in Shanxi Province Cancer Hospital from January 2020 to December 2021 were selected, and the clinical data of the patients were retrospectively analyzed. The mutation status of KRAS, NRAS and BRAF V600E genes were detected in the paraffin specimens of surgically-resected tissues by direct sequencing. The mutation rates of KRAS, NRAS and BRAF V600E were compared among patients with different clinicopathological characteristics.Results:The mutation rates of KRAS, NRAS and BRAF V600E in 217 patients with colorectal cancer were 48.4% (105/217), 4.1% (9/217) and 3.7% (8/217), of which 1 patient (0.5%) had both KRAS and NRAS mutations. NRAS gene mutation was not correlated with gender, age, tumor size, tumor location, pathological type, degree of differentiation, depth of invasion, lymph node metastasis, distant metastasis, TNM stage, hemangioma thrombus/nerve invasion (all P>0.05); KRAS mutation rate in patients ≥ 60 year old was higher than that in patients < 60 year old [55.3% (63/114) vs. 40.8% (42/103), χ2 = 4.55, P = 0.033),and there was no correlation between KRAS gene mutation and other clinicopathological features (all P > 0.05); the mutation rate of BRAF V600E gene in colorectal cancerpatients with distant metastasis was higher than that in patients without distant metastasis [16.7% (4/24) vs. 2.1% (4/193), P = 0.006], and there was no correlation between BRAF V600E gene mutation and other clinicopathological features (all P > 0.05). Conclusions:Older colorectal cancer patients may be prone to KRAS gene mutation, and the BRAF V600E gene mutation rate is higher in patients with distant metastasis, and there is no correlation between NRAS gene mutation and clinicopathological characteristics.

Evaluación de la metilación del DNA en tejidos de cáncer colorrectal fijados en formalina y embebidos en parafina (FFPE) / Evaluation of DNA methylation in formalin-fixed, paraffin embedded colorectal cancer tissues (FFPE)

Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.

Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.

Cytoplasmic-membrane egfr predicts expanded ras mutation status in colorectal carcinomas? / Egfr membrano-citoplasmático prediz mutação ras expandida no carcinoma colorretal?

ABSTRACT Background: Inhibitors of the epidermal growth factor (EGFR) represent an effective therapeutic option for patients with metastatic colorectal carcinoma, free of activating mutations in KRAS and NRAS. However, the research of mutations is of high cost and scarcely accessible. The expression of the EGFR by immunohistochemistry predicting the mutation status of the expanded RAS (KRAS and NRAS), may allow treatment by a diagnostic method less costly and more accessible. Aim: Investigate the correlation between the clinical-pathological data, the cytoplasmic-membrane expression of the EGFR and the mutational status of the expanded RAS. Method: A total of 139 patients with colorectal carcinoma from the archives of Instituto Goiano de Oncologia e Hematologia were evaluated. Results: Mutation of the expanded RAS was detected in 78 (56.1%) cases. The EGFR expression was stratified in 23 (16.5%) "positive", 49 (35.2%) "negative" and 67 (48.2%) "uncertain". No significant correlation was found between the mutational status of the RAS and the EGFR expression in comparison to age, gender, location, histological type, histological grade and stage. From 23 "positive" cases, 21 (91.3%) showed wild-type RAS gene, and 49 "negative", 41 (83.7%) presented mutation, resulting in a strong association between EGFR "positive", "negative" groups and the mutational status of the RAS (p<0.001), with 86.1% of accuracy. Conclusions: The cytoplasmic-membrane analysis of the EGFR expression stratified into "positive", "negative" and "uncertain" predicts mutational status of the RAS in 51.7% of the cases (p<0.001), with 86.1% of accuracy.

RESUMO Racional: Inibidores do fator de crescimento epidermal (EGFR) representam opção de terapia efetiva para o câncer colorrectal metastático, na ausência de ativação de mutações KRAS e NRAS. Entretanto, a pesquisa de mutações é cara e pouco acessível. A expressão de EGFR por imuno-histoquímica predizendo o status mutacional do RAS expandido (KRAS e NRAS) poderia permitir o tratamento por método diagnóstico menos caro e mais acessível. Objetivo: Investigar a correlação entre os dados clinicopatológicos, a expressão de EGFR na membrana citoplasmática e o status mutacional do RAS expandido. Método: Estudo retrospectivo de acurácia envolvendo 139 pacientes com carcinoma colorretal. Resultado: A mutação do RAS expandido foi detectada em 78 (56,1%) casos. A expressão de EGFR foi estratificada em 23 (16,5%) casos "positivos", 49 (35,2%) casos "negativos" e 67 (48,2%) "duvidosos". Não houve correlação significante entre o status mutacional do RAS e a expressão de EGFR em relação a idade, gênero, local do tumor, tipo histológico, grau histológico e estádio clínico. Em 23 casos "positivos", 21 (91,3%) mostraram gene RAS tipo selvagem, e em 49 "negativos", 41 (83,7%) apresentaram mutação, resultando em forte associação entre grupos EGFR "positivo" ou "negativo" e o status mutacional do RAS (p<0.001), com 86,1% de acurácia. Conclusão: A análise da expressão de EGFR na membrana citoplasmática estratificada em "positivo", "negativo" e "duvidoso" prediz o status mutacional do RAS em 51,7% dos casos (p<0.001), com 86,1% de acurácia.

Combined detection of KRAS, NRAS, BRAF and PIK3CA mutations in the plasma and tumor tissues of colorectal cancer patients / 中华病理学杂志

Objective@#To analyze the concordance of KRAS, NRAS, BRAF and PIK3CA gene mutations detected in plasma and matched tumor tissues in colorectal cancer patients, in order to provide good evidences to support plasma could be a potential surrogate of tumor tissue for gene mutation test.@*Methods@#One hundred and seventy-five cases of colorectal cancer were collected at the First Hospital of Jilin University, from October 2016 to October 2017.There were 101 males and 74 females, their ages ranged from 28 to 85 years,with median age of 59 years. The KRAS, NRAS, BRAF and PIK3CA gene mutations in the plasma and paired tumor specimens of all patients were detected by next generation sequencing.@*Results@#The results of tissue samples test were gold standard. Comparison of the four genes showed that concordance rates between plasma and tissue samples were 81.1%(Kappa=0.543), 99.4%(Kappa=0.886), 99.4% (Kappa=0.886) and 97.7%(Kappa=0.714) respectively for KRAS, NRAS, BRAF and PIK3CA. The plasma detection rates of these genes were related to tumor stage(P=0.001), but not to gender(P=0.468) and age(P=1.000) of patients.@*Conclusions@#The study shows a high concordance of KRAS, NRAS, BRAF and PIK3CA gene mutations in plasma against mutation status in tumor tissue. In colorectal cancer, tumor tissue remains the best specimen for gene detection. However, patients from tumor tissue specimens cannot be obtained, especially those with advanced metastases, plasma can be used instead of tissue to detect the mutation status of KRAS, NRAS, BRAF and PIK3CA to guide targeted therapy.

KRAS mutations analysis in mucinous epithelial lesions of the endometrium / 中华病理学杂志

Objective@#To investigate the frequency of KRAS mutation in mucinous epithelial lesions of the endometrium, and analyze the correlation between KRAS mutation and the clinicopathologic features.@*Methods@#The cohort included forty-three cases of mucinous epithelial lesions of the endometrium selected from July 2015 to October 2017 from Beijing Obstetrics and Gynecology Hospital, and 22 control cases. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue sections. Polymerase chain reaction amplification for KRAS exons 2 and 3 was performed, followed by sequencing using capillary electrophoresis. The Fisher exact test was used to compare the prevalence of KRAS mutation among the different groups.@*Results@#The patients′age ranged from 33 to 77 years [mean (55.12±9.34) years, median 55 years]. None of the eight cases of endometrial hyperplasia with mucinous differentiation without atypia showed KRAS mutation. The frequency of KRAS mutations was 1/10 in endometrial atypical hyperplasia, 1/12 in endometrioid carcinoma, 4/11 in endometrial atypical hyperplasia with mucinous differentiation (EAHMD), 6/15 in endometrioid carcinoma with mucinous differentiation (ECMD) and 8/9 in mucinous carcinoma (MC), respectively. The differences were statistically significant between MC versus EC (P<0.01) and MC versus ECMD (P<0.05).@*Conclusion@#The high frequency of KRAS mutation in EAHMD, ECMD and MC indicates that KRAS mutational activation is implicated in the pathogenesis of endometrial mucinous carcinoma.

Correlation between K-ras gene mutation and clinicopathological characteristics and prognosis of colorectal cancer patients with different primary sites / 肿瘤研究与临床

Objective To analyze the correlation between K-ras gene mutation and clinicopathological characteristics and prognosis of colorectal cancer patients with different primary sites.Methods The clinical and pathological records of 69 patients who were pathologically confirmed as colorectal cancer and tested K-ras gene status at Wuxi People's Hospital Affiliated to Nanjing Medical University between May 2007 and August 2017 were adopted.The correlation between clinicopathological characteristics and prognosis of colorectal cancer patients with different primary sites and K-ras gene mutation status were retrospectively analyzed.And the patients were visited to adopt the prognosis data and perform the Kaplan-Meier survival analysis.Results The K-ras mutation rate was 50.7％(35/69),including 40.0％(12/30)in left-side colon cancer,73.3％(11/15)in right-side colon cancer and 50.0％(12/24)in rectal cancer.The mutation rate of K-ras gene in patients ≥ 60 years old [61.5％(24/39)vs.36.7％(11/30),χ2= 4.197,P = 0.041] or serum CA19-9 raising up abnormally was conspicuously high [65.6％(19/29)vs.37.5％(15/40),χ 2= 5.486,P =0.019].Other clinicopathological characteristics,such as gender,lesion location,histological classification,TNM stage,serum CEA expression,clinical features had no correlation with K-ras gene mutation status(all P＞ 0.05).And no relationship was found between prognosis and overall survival of colorectal cancer patients with different primary sites and K-ras gene mutation status(χ 2= 0.001,P = 0.997; χ 2= 0.583,P =0.445).In general,the 5-year survival rate of left-side colon cancer patients was highest(76.9％),followed by rectal cancer(69.7％),and right-side colon cancer was lowest(31.3％).The primary site of colorectal cancer was related to the overall survival of patients(χ2=11.004,P =0.004).Conclusions K-ras gene mutation in colorectal cancer is closely related to age and serum CA19-9 levels of the patients.The prognosis of left-side colon cancer is best,rectal cancer second,and right-side colon cancer poorest.Whether K-ras gene mutation is the prognostic factor of colorectal cancer is not clear.Testing the K-ras gene status and serum tumor index expression,distinguish the primary site and age group will provide the theory basis and promote the clinical targeted therapy and improve the survival of colorectal cancer patients.

Research on ligase-ELISA method for detecting K-ras gene mutations / 重庆医学

Objective To research a simple and sensitive K-ras gene mutations detection method in order to be suitable for the routine mutation detection.Methods The corresponding detection locus oligonucleotide probe was designed.By the connection,amplification,labeling and ELISA reaction in probe,the mutation locus genotype was determined by the ELISA reaction detection value.With the six point mutations of G12S,G12R,G12C,G12D,G12A and G12V in 12 codons of K-ras gene as the detection objects,the plasma circulation DNA sample in 72 cases of lung cancer was detected,then the results were compared with those obtained by the direct sequencing.Results Three samples were identified as the G12S,G12R and G12A mutatins by the established method.But no K-ras mutations were detected in the samples by using the direct sequencing,indicating that the direct sequencing had lower sensitivity and was not suitable for the mutation detection of heterogeneous samples such as circulating DNA.Conclusion The simple and sensitive K-ras gene mutation detection method is established and can conduct the routine mutation detection for the heterogeneous samples.

Effect of proliferation of triptolide on MV4-11 cells and its mechanism research associated with RAS/ERK/MAPK / 中国医师杂志

Objective To explore the effect of proliferation and apoptosis,and research its mechanism associated with RAS/extracellular signal regulated kinase/mitogen-activated protein kinase (RAS/ ERK/MAPK) in Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation acute lymphocytic leukemia cell line MV4-11 cells treated by triptolide (TP).Methods MV4-11 cells were respectively treated by triptolide with diverse concentrations and different times.The proliferation inhibition rate was measured by methyl thiazolyl tetrazolium,the apoptosis rate was detected by flow cytometry,the mRNA expressions of FLT3,RAS,ERK,forkhead box protein M1 (FOXM1),and c-Myc were analyzed by realtime fluorescent quantitative polymerase chain reaction (PCR).Results The proliferation inhibition rate markedly increased in a dose-time dependent manner after MV4-11 cells were treated by TP.After cells were treated with 10,and 20 nmol/L TP,respectively,the apoptosis rates at 48 h were (17.30 ± 0.56) ％,(35.77 ±0.55)％,and those at 72 h were (49.83 ±0.45)％,(68.90 ±0.75)％ correspondingly.PCR data showed that the mRNA level of FLT3 mRNA decreased obviously,and that of RAS,ERK,FOXM1,and c-Myc also decreased.Conclusions Triptolide could significantly inhibit the MV4-11 cells proliferation and induce cell apoptosis,and its mechanism might be through inhibiting the expression of related genes on RAS/ERK/MAPK signaling pathway.

Síndrome cardio-facio-cutáneo: reporte de caso / Cardio-facio-cutaneous syndrome: a case report

Resumen: el síndrome cardio-facio-cutáneo es una entidad clínica y genéticamente heterogénea, perteneciente a un grupo de síndromes conocidos como RASopatías.Este trastorno es de baja prevalencia, con alrededor de 200 a 300 casos en el mundo, e incluye entre sus manifestaciones clínicas rasgos faciales dismórficos, defectos cardíacos y alteraciones cutáneas. Los hallazgos fenotípicos del síndrome cardio-facio-cutáneo que se comparten con otros síndromes y la ausencia de criteriosdiagnósticos o signos patognomónicos lo convierten en un reto diagnóstico. En este manuscrito se presenta un caso confirmado de síndrome cardio-facio-cutáneo por estudios de genética molecular en una paciente de siete años de edad, mediante el cual se exponen las principales características de esta condición.

Abstract: The cardio-facio-cutaneous syndrome is a clinically and genetically heterogeneous disorder, belonging to a group of syndromes known as RASopathies. This condition has a low prevalence, with around of 200 to 300 cases in the world, and includes dysmorphic facial features, heart defects, and skin abnormalities among its clinical manifestations. The phenotypic findings of cardio-facio-cutane1ous syndrome that are shares with other syndromes and the absence of diagnostic criteria or pathognomonic signs make it a diagnostic challenge. Here its present a confirmed case of cardio-facio-cutaneous syndrome by molecular genetic studies in one seven years old patient, through which are exposed the main characteristics of this condition.

Syndrome in question

Costello syndrome (CS) is a rare genetic disorder, first described by Costello in 1971, caused by mutations in the HRAS proto-oncogene. Clinical findings include facial dysmorphism, skin disorders, cognitive impairment, cardiac and musculoskeletal defects. There is an increased risk of malignancies in these patients, due to the proto-oncogene mutation, and also sudden death secondary to heart disease. We report a case with characteristic phenotype, highlighting the peculiar skin changes.

Quantitative detection and the significance of K-ras12 and K-ras13 mutations in peripheral blood of the population with high risk of pancreatic cancer / 中华消化杂志

Objective To explore the clinical significance of the detection of K-ras12 and K-ras13 mutations in the peripheral blood of population with high risk group of pancreatic cancer.Methods From May 2010 to November 2011,a total of 160 patients with clear diagnosis and high risk of pancreatic cancer were collected.Among those patients,36 cases were finally diagnosed as pancreatic cancer,36 cases were chronic pancreatitis (CP),six cases were autoimmune pancreatitis (AIP),16 cases were other malignant tumor,13 cases were pancreatic benign tumor,13 cases were bile duct stones and 40 cases were other benign diseases.Fasting peripheral venous blood was collected in all selected candidates.The condition of K-ras12 and K-rasl3 mutation was detected by peptide nucleic acid-mediated real time fluorescen quantitative polymerase chain reaction.Mann-Whitney U test was performed to compare the mutation between the two groups.Chi-square test was used to compare the rates.According to the receiver operator characteristic (ROC) curve,the mutation positive decision value and the mutation positive rate of K-ras12 and K-ras13 were calculated.Results There were significant differences in K-ras12 mutation between pancreatic cancer group (1154(2207)) and other benign diseases (476(973)),CP group (446(808)),bile duet stones group (357(568)) (U=502,446 and 117,all P＜0.05).There were no significant differences in K-ras13 mutation between pancreatic cancer group and other benign diseases,CP group,bile duct stones group (all P＞0.05).When the mutation positive decision value of K-ras12 was set as mutation copy over 1000,the positive mutation rate of pancreatic cancer and non-pancreatic cancer was 55.6％ (20/36) and 25.0％ (31/124),respectively.When the area under the ROC curve of K-ras12 was 0.664,there was differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.003).When the area under the ROC curve of K-ras13 was 0.522,there was no differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.695).There was significant difference in K-ras13mutation between smokers and non-smokers (943 (1510) and 571 (964)).Conclusion K-ras12mutation in peripheral venous blood may be used for pancreatic cancer screening.

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip / 中华检验医学杂志

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation.Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results.Results The sensitivity of liquid chip technology detection was 10％-20％,higher than the traditional sequencing method by 1％.Average CV value was 4％-15％ and showed good repeatability.5 K-ras mutations in 100 patients (5％) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Glyl2Val and 2 Gly12Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing.Conclusions The novel detection method of K-ras mutations based PCRLDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.

Genotyping of K-ras exon 2 codons 12 and 13 mutations in colorectal cancer by pyrosequencing / 中华检验医学杂志

ObjectiveTo investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived fromclinicalformalin-fixed andparaffinembedded(FFPE)tissues.ResultsCancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％(12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.

Quantitative detection of K-ras gene mutation in samples from endoscopic ultrasound-guided fine-needle aspiration for pancreatic cancer / 中华消化内镜杂志

Objective To investigate the diagnostic value of a quantitative detection of K-ras mutation in samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA)of pancreatic cancer.Methods Samples taken by EUS-FNA from 53 pancreatic occupying lesions were collected, and the copies of wild-type and mutated K-ras gene was measured by PNA-clamping real-time quantitative PCR. The results were analyzed with refer to cytological findings to evaluate its clinical values. Results According to cytological finding, a total of 37 cases were diagnosed as pancreatic cancer, and 16 were non-malignant lesions. Kras mutation was detected in 83.8% of cancer cases, and 18. 8% of non-cancer cases, which was significantly different ( P ＜0. 05 ). Sensitivities of cytology and K-ras examination were 59. 5% and 83.8%, respectively, while that of combination of cytology and K-ras examination was 89. 2%. Conclusion Quantitative analysis of the mutant K-ras gene in samples taken by EUS-FNA is a useful tool for diagnosing the pancreatic carcinoma.

Inducing apoptosis effect by 188Re-k-ras-AGPNA on pancreatic cancer cells and biodistribution characteristics in nude mice bearing xenografts / 中华核医学杂志

Objective To investigate the depressing effect of antigene peptide nucleic acid (AGPNA)on the k-ras gene expression of human pancreatic cancer Patu8988 cells, the inducing apoptosis effect on Patu8988 cells, and the biodistribution characteristics in nude mice bearing xenografts using 188Re-k-ras-AGPNA.Methods The expression level of k-ras mRNA and the expression ratio of k-ras protein in Patu8988 cells transfected with AGPNA was measured by RT-PCR and flow cytometry ,respectively. The degree of cellular apoptosis 3 to 5 d after treating Patu8988 cells with 188Re-k-ras-AGPNA or 188ReO4- was determined by flow cytometry. For biodistribution study, 58 nude mice bearing Patu8988 cell xenografts were divided into two groups: intratumoral injection of 188Re-k-ras-AGPNA (Group A) and 188ReO4- (Group B). At different time points, the mice were sacrificed and organs of interest were excised, weighted and counted by a gamma counter. The organ uptake was calculated as a % ID/g and the absorbed doses of organs were calculated. One-way analysis of variance was used. Results After transfected with 1 nmol/ml AGPNA, the k-ras mRNA gray scale ratio and the expression ratio of k-ras protein were 1.00 ± 0.39 and (15.05 ± 5.07)%, respectively. They were significantly lower than those of the control group with 1.86 ± 0.07 and (24. 38 ± 5.40) % (F = 2. 545, 5. 327, P<0. 05). At 4 and 5 d after treatment in Group A, float cells' apoptosis ratios were (26.30 ± 7.45) % and (27.90 ± 10. 38) %, respectively. Tumors were the major distribution site in Group A with uptake of (37.47 ±21.31), (35.96 ±7.80) and (15.46 ±4.93) %lD/g at 1 h, 1 d and 7 d after intra-tumor injection, respectively. The absorbed dose of tumor was 15 569 mGy/MBq. Condusions Transfection with k-ras-AGPNA on Patu8988 cells may inhibit k-ras expression at mRNA and protein expression level, and 188Re-k-ras-AGPNA can induce apoptosis of Patu8988 cells.Tumor is the major distribution site in nude mice bearing human pancreatic cancer xenografts after intratumoral injection of 188Re-k-ras-AGPNA.

K-ras mutations in colorectal cancer at different stages / 肿瘤

Objective:To study the alteration of K-ras mutations in different stages of colorectal cancer(CRC) and its influence on the progression of CRC. Methods:The 20 paraffin-embedded tissues, including primary foci, metastatic lymph nodes, remoter metastatic foci, colorectal adenoma, and normal colorectal tissues, were collected from 20 patients with colorectal cancer. The sequence of PCR-amplified products were analyzed. Results:The wild K-ras gene was expressed in normal colorectal tissues. The mutation frequency of K-ras gene was 20.0% (4/20) in colorectal adenoma, 30.0% (6/20) in primary foci, 25.0% (5/20) in metastatic lymph nodes, and 30% (6/20) in remote metastatic lesions. In the samples with K-ras mutations, the consistency of the types of K-ras mutations between primary foci and colorectal carcinoma, lymph node metastatic lesions, remote metastatic lesions was 0.0%(0/4), 40.0%(2/5), and 50.0%(3/6), respectively.Conclusion:The colorectal adenoma, metastatic lymph nodes and remote metastatic lesions were not suited for K-ras analysis as routine samples in clinical practice. If the samples of primary lesions were not available, the detection results of metastatic lymph nodes and remote metastatic remote lesions will provide some reference values. K-ras gene had several different mutations in the progression of CRC.