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PURPOSE: Radioiodine (RAI) therapy remains the gold-standard approach for distant metastatic differentiated thyroid cancer (TC). The main objective of our work was to identify the clinical and molecular markers that may help to predict RAI avidity and RAI therapy response of metastatic lesions in a cohort of papillary thyroid cancer (PTC) patients. METHODS: We performed a retrospective analysis of 122 PTC patients submitted to RAI therapy due to distant metastatic disease. We also analysed, through next-generation sequencing, a custom panel of 78 genes and rearrangements, in a smaller cohort of 31 metastatic PTC, with complete follow-up, available RAI therapy data, and existing tumour sample at our centre. RESULTS: The most frequent outcome after RAI therapy was progression of disease in 59.0% of cases (n = 71), with median estimate progression-free survival of 30 months. RAI avidity was associated with PTC subtype, age and stimulated thyroglobulin at first RAI therapy for metastatic disease. The most frequently altered genes in the cohort of 31 PTC patients' primary tumours were RAS isoforms (54.8%) and TERT promoter (TERTp) (51.6%). The presence of BRAF p.V600E or RET/PTC alterations was associated with lower avidity (p = 0.012). TERTp mutations were not associated with avidity (p = 1.000) but portended a tendency for a higher rate of progression (p = 0.063); similar results were obtained when RAS and TERTp mutations coexisted (p = 1.000 and p = 0.073, respectively). CONCLUSIONS: Early identification of molecular markers in primary tumours may help to predict RAI therapy avidity, the response of metastatic lesions and to select the patients that may benefit the most from other systemic therapies.

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BACKGROUND/AIM: Complete clinical response in rectal cancer after neoadjuvant chemo-radiotherapy is challenging. Indeed, indication to surgery vs. "watch and wait" is a debate due the poor predictive value of restaging exams in order to identify a pathological complete response (pCR). Improving the knowledge on mutational pathways such as MAPK/ERK could be helpful in assessing the real impact of disease on prognosis and in choosing the best therapeutic target. This study aimed to evaluate the significance of biomolecular parameters as prognostic factors in patients undergoing radical surgery after chemo-radiotherapy. PATIENTS AND METHODS: A retrospective analysis was performed including 39 patients who had undergone radical surgery after neoadjuvant chemo-radiotherapy for rectal adenocarcinoma stage II-III through additional evaluation of the following biomolecular markers on surgical specimens: exons 2, 3 and 4 of the KRAS and NRAS genes and exon 15 of BRAF by pyrosequencing. Kaplan-Meier survival curves were plotted to evaluate the association of pathologic response and RAS status with progression-free survival (PFS) and overall survival (OS). The log-rank test was used to assess statistical differences among the survival curves. RESULTS: Data analysis showed RAS mutation in 15 patients (38.46%). pCR was achieved in seven patients (18%), including only two RAS mutation cases. The distribution of evaluated variables was homogeneous in the two groups based on pathological response. The Kaplan-Meier curve showed poor outcomes in OS and PFS in patients with RAS mutation (p=0.0022 and p=0.000392, respectively), but no significant differences based on pathological response for both OS and PFS. CONCLUSION: RAS mutation seems to be related to poor prognosis and increased risk of recurrence in rectal cancer patients undergoing radical surgery after chemo-radiotherapy.

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Codon usage influences gene expression distinctly depending on the cell context. Yet, the importance of codon bias in the simultaneous turnover of specific groups of protein-coding genes remains to be investigated. Here, we find that genes enriched in A/T-ending codons are expressed more coordinately in general and across tissues and development than those enriched in G/C-ending codons. tRNA abundance measurements indicate that this coordination is linked to the expression changes of tRNA isoacceptors reading A/T-ending codons. Genes with similar codon composition are more likely to be part of the same protein complex, especially for genes with A/T-ending codons. The codon preferences of genes with A/T-ending codons are conserved among mammals and other vertebrates. We suggest that this orchestration contributes to tissue-specific and ontogenetic-specific expression, which can facilitate, for instance, timely protein complex formation.

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BACKGROUND: Local sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14. RESULTS: Molecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts. CONCLUSIONS: We conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development.

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Overexpression of normal Ras and its aberrant CpG island methylation in the promoter regions have been shown to direct cells for uncontrolled abnormal growth and bladder tumor formation and therefore, fetched recent attention as a marker of diagnosis and prognosis to predict the biological behavior of urothelial carcinoma of bladder (UCB). Methylation pattern at CpG islands of the promoter regions of rat sarcoma (Ras) gene homologues namely Kristen-Ras (K-Ras), Harvey (H-Ras), and Neuroblastoma (N-Ras) were examined by methylation specific polymerase chain reaction (MSP). Real time-quantitative polymerase chain reaction (RT-qPCR) was done to determine transcriptomic expressions of these Ras isoforms in the prospective series of 42 NMIBC (non-muscle invasive bladder cancer) and 45 MIBC (muscle invasive bladder cancer) biopsies. CpG loci in H-Ras and K-Ras were observed to be more hypomethylated in MIBC, whereas more hypomethylation in N-Ras was noted in NMIBC. Strong association of hypomethylation index with tumor stage, grade, type and size validate them it as marker of diagnosis in UCB patients. Differential overexpression of H-Ras, N-Ras and K-Ras genes in NMIBC and MIBC and their association with patients' demographics identify them as important diagnostic markers in pathogenesis of UCB. Given the reported ability of promoter hypomethylation to activate Ras expression, correlation studies examined positive significant association between hypomethylation index and expression. Study concludes that promoter hypomethylation of N-Ras and K-Ras could be a potential confounder of their increased expression in NMIBC. Biological significance of simultaneous presence of higher expression and promoter hypomethylation of Ras gene isoforms in MIBC is difficult to resolve in a given cohort of patients.

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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.

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Abstract Background: The presence of clinically relevant mutations in KRAS and NRAS genes determines the response of anti-epidermal growth factor receptor antibody therapy for metastatic colorectal cancer (mCRC). The only quantitative polymerase chain reaction (qPCR)-based diagnostic tests approved by the Food and Drug Administration (FDA) screen merely for mutations in codons 12 and 13 of KRAS. Objective: The objective of the study was to study the frequency of clinically relevant mutations in KRAS and NRAS genes that are not included in FDA-approved qPCR tests. Methods: Formalin-fixed paraffin-embedded tumor specimens from 1113 mCRC Mexican patients from different health institutions across the country were analyzed by Sanger sequencing for KRAS mutations in exons 2, 3, and 4. Furthermore, 83 were analyzed in exons 2, 3, and 4 of NRAS. Results: From the specimens tested for KRAS, 33.69% harbored a mutation. From these, 71.77% were in codon 12 and 27.69% in codon 13 (both located in exon 2). Codons 59 (exon 3) and 146 (exon 4) accounted for the remaining 0.54%. From the 83 specimens, in which NRAS was analyzed, three mutations were found in codon 12 (3.61%). Approximately 6% of RAS mutated specimens would have been falsely reported as RAS wild type if an FDA-approved qPCR diagnostic test had been used. Conclusions: While these kits based on qPCR can be very practical and highly sensitive, their mutation coverage ignores mutations from poorly genetically characterized populations.

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BACKGROUND: Mutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer. Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder (UCB). AIM: To determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome. METHODS: This study was performed to determine the mutational spectrum at the hotspot regions of H-Ras, K-Ras and N-Ras genes by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing followed by their clinical impact (if any) by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients. RESULTS: None of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene; codon 61 of N-Ras gene and codons 12, 13 of K-Ras gene by PCR-RFLP. Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras, N-Ras and K-Ras genes in the tumor and control bladder mucosal specimens. CONCLUSION: Our findings on the lack of mutations in H-Ras, K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression, inherent genetic susceptibility, tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

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Members of the rat sarcoma (RAS) gene family belong to the most frequently mutated genes that drive pathogenesis and therapy response. As the discovery of their malignant potential dates back more than three decades, cellular mutated RAS genes and their products belong to the best characterized cancer genes. Despite urgent clinical needs, RAS therapies are still elusive and limited to preclinical studies. However, very recently, novel and promising approaches have become a reality in clinical applications and trials. In the near future, interesting therapeutic options will emerge that are capable of targeting "undruggable" RAS. This will be even more important as the detection of RAS mutations has already been an integral part of routine molecular diagnostics for many years.

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To reveal novel lncRNAs and explore how could lncRNA affect the ER/PR positive type breast cancer, 16 different lncRNA transcriptomes (8 breast cancer tissues and 8 normal breast tissues) were successfully sequenced. In total, 8,954 high quality lncRNAs, including 5,516 lncRNAs reported in the previous studies and 3,438 novel lncRNAs, were annotated. The highest expressed lncRNAs were MALAT1, SCARNA10, RP11-206M11.7 and NEAT1, and the highest expressing mRNAs were RPL19, SCGB2A2, FTL and TMSB4 × . Of the 615 differentially expressed lncRNAs, 323 showed up regulated (P < 0.05) expression patterns in breast cancer, and 292 showed down regulated expression patterns. Of the 8,954 genes, 5,516 genes were upregulated in breast cancer, and 3,438 were downregulated. In total, the targets of 238 lncRNAs were confirmed by two lncRNA target prediction programs. Within these genes, Ras responsive element binding protein 1, Ras association domain family member 6, Ras association domain family member 8, Ras protein specific guanine nucleotide releasing factor 1and other 10 different Ras associated different expressed genes were predicted as targets of lncRNAs. These different expressed lncRNAs which could regulate the Ras gene families and ECM pathway may be another mechanism why the expression pattern of Ras genes changed in breast cancer. All these cancer-related genes (Ras genes) were annotated as targets of lncRNAs in the breast cancer transcriptome may provide us with a new way to understand the occurrence and development of breast cancer.

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Epidermal nevi are hamartomatous lesions derived from the epidermis and/or adnexal structures of the skin; they have traditionally been classified according to their morphology. New variants have been described in recent years and advances in genetics have contributed to better characterization of these lesions and an improved understanding of their relationship with certain extracutaneous manifestations. In the second part of this review article, we will look at nevi derived from the adnexal structures of the skin and associated syndromes.

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Follicular-patterned tumors of the thyroid gland are characterized by a predominantly follicular growth pattern. They frequently harbor RAS mutations, not BRAF mutations. Technological advances in molecular testing have discovered novel RAS-type mutations. However, clinical significance of these mutations remains unknown. We investigated the prevalence and clinical impact of mutations of BRAF, NRAS, HRAS, KRAS, EZH1, EIF1AX, and TERT genes by Sanger sequencing in a series of 201 follicular-patterned thyroid tumors including follicular adenoma (n = 40), Hürthle cell adenoma (n = 54), noninvasive follicular thyroid neoplasms with papillary-like nuclear features (n = 50), follicular thyroid carcinoma (n = 40), Hürthle cell carcinoma (n = 10), and poorly differentiated thyroid carcinoma arising in a well-differentiated follicular neoplasm (n = 7), and 120 classic papillary carcinoma. Two hotspots of EZH1 mutations were only found in RAS-negative follicular-patterned tumors. EZH1 mutations were detected in 3% of follicular adenoma and in 20% of Hürthle cell adenoma, and one minimally invasive Hürthle cell carcinoma. Thyroid tumors with EZH1 mutations reported in the literature were benign in most cases. Otherwise, they were minimally invasive or noninvasive cancer. EIF1AX mutation was found in one follicular adenoma. We confirmed the presence of RAS mutations and BRAF K601E mutation in benign, borderline, and malignant follicular-patterned tumors. No BRAF V600E was found in all follicular-patterned tumors. This study also confirmed the occurrence of TERT promoter mutations in high-risk thyroid cancers. These genetic markers can be used for the diagnostic purpose and risk stratification of thyroid nodules.

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PURPOSE: Cetuximab demonstrates improved efficacy outcomes in patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS exon 2. Resistance to cetuximab is mediated by activating less frequent mutations in the RAS genes beyond KRAS exon 2. We performed extended RAS Mutational analysis using a high-throughput genotyping platform (OncoMap) and evaluated extended RAS analysis for predicting cetuximab efficacy in patients harboring wild-type KRAS exon 2 tumors following Sanger sequencing. MATERIALS AND METHODS: Extended RAS analysis was performed on 227 wild-type KRAS exon 2 mCRC patients who received cetuximab as salvage treatment using OncoMap ver. 4.0. Targeted genes included exon 2, exon 3, and exon 4, both in KRAS and NRAS, and included BRAF exon 15. We assessed efficacy by the new RAS mutation status. RESULTS: The OncoMap detected 57 additional mutations (25.1%): 25 (11%) in KRAS exon 2 and 32 (14.1%) beyond KRAS exon 2. Survival differences were observed after dividing patients into the wild-type RAS group (n=170) and mutant RAS group (n=57) using OncoMap. Progression-free survival was 4.8 months versus 1.8 months (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.32 to 0.61), and overall survival was 11.9 months versus 8.4 months (HR, 0.65; 95% CI, 0.47 to 0.88). CONCLUSION: Sanger sequencing is not sufficient for selecting candidates for cetuximab treatment. High-throughput extended RAS genotyping is a feasible approach for this purpose and identifies patients who might benefit from cetuximab treatment.

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PURPOSE: Cetuximab demonstrates improved efficacy outcomes in patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS exon 2. Resistance to cetuximab is mediated by activating less frequent mutations in the RAS genes beyond KRAS exon 2. We performed extended RAS Mutational analysis using a high-throughput genotyping platform (OncoMap) and evaluated extended RAS analysis for predicting cetuximab efficacy in patients harboring wild-type KRAS exon 2 tumors following Sanger sequencing. MATERIALS AND METHODS: Extended RAS analysis was performed on 227 wild-type KRAS exon 2 mCRC patients who received cetuximab as salvage treatment using OncoMap ver. 4.0. Targeted genes included exon 2, exon 3, and exon 4, both in KRAS and NRAS, and included BRAF exon 15. We assessed efficacy by the new RAS mutation status. RESULTS: The OncoMap detected 57 additional mutations (25.1%): 25 (11%) in KRAS exon 2 and 32 (14.1%) beyond KRAS exon 2. Survival differences were observed after dividing patients into the wild-type RAS group (n=170) and mutant RAS group (n=57) using OncoMap. Progression-free survival was 4.8 months versus 1.8 months (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.32 to 0.61), and overall survival was 11.9 months versus 8.4 months (HR, 0.65; 95% CI, 0.47 to 0.88). CONCLUSION: Sanger sequencing is not sufficient for selecting candidates for cetuximab treatment. High-throughput extended RAS genotyping is a feasible approach for this purpose and identifies patients who might benefit from cetuximab treatment.

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Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine.

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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.

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BACKGROUND: Tumour shrinkage (TS) increases the possibility of resection in metastatic colorectal cancer (mCRC) and may improve tumour-related symptoms. Here we report prespecified secondary response-related end-points and exploratory TS/resection outcomes for patients with RAS wild-type (WT) tumours (no mutations in KRAS/NRAS exons 2/3/4) from the PRIME study (NCT00364013). METHODS: PRIME was a randomised phase 3 study comparing first-line panitumumab+FOLFOX4 versus FOLFOX4 in mCRC patients. Tumour response analyses were conducted to compare response rates and their impact on survival outcomes. RESULTS: Overall, 505 patients had RAS WT mCRC. More patients receiving panitumumab+FOLFOX4 versus FOLFOX4 had ⩾30% (59% versus 38%; P<0.001) or ⩾20% (72% versus 57%; P<0.001) TS at week 8 (early TS); consistent TS benefits were observed over the first ∼40weeks of treatment. Objective response rate (P=0.003), duration of response (P=0.0027), depth of response (P=0.0149), progression-free survival (PFS; P=0.0015) and overall survival (OS; P=0.0057) were improved in the panitumumab+FOLFOX4 group. Both early TS and resection were associated with improved PFS and OS. 2-year OS rates for patients who did (n=64) versus did not (n=441) undergo resection were 88% versus 40%; 2-year OS rates for patients who did (n=45) versus did not (n=460) undergo complete resection were 96% versus 41%. CONCLUSIONS: More patients receiving panitumumab+FOLFOX4 versus FOLFOX4 had ⩾30% or ⩾20% TS at week 8; PFS and OS were also improved with panitumumab+FOLFOX4. The clinical value of achieving early TS in mCRC warrants further investigation.

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The Japanese guidelines for the testing of KRAS mutations in colorectal cancer have been used for the past 5 years. However, new findings of RAS (KRAS/NRAS) mutations that can further predict the therapeutic effects of anti-epidermal growth factor receptor (EGFR) antibody therapy necessitated a revision of the guidelines. The revised guidelines included the following five basic requirements for RAS mutation testing to highlight a patient group in which anti-EGFR antibody therapy may be ineffective: First, anti-EGFR antibody therapy may not offer survival benefit and/or tumor shrinkage to patients with expanded RAS mutations. Thus, current methods to detect KRAS exon 2 (codons 12 and 13) mutations are insufficient for selecting appropriate candidates for this therapy. Additional testing of extended KRAS/NRAS mutations is recommended. Second, repeated tests are not required for the detection; tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing. Evaluating RAS mutations prior to anti-EGFR antibody therapy is recommended. Third, direct sequencing with manual dissection or allele-specific PCR-based methods is currently applicable for RAS mutation testing. Fourth, thinly sliced sections of formalin-fixed, paraffin-embedded tissue blocks are applicable for RAS mutation testing. One section stained with H&E should be provided to histologically determine whether the tissue contains sufficient amount of tumor cells for testing. Finally, RAS mutation testing must be performed in laboratories with appropriate testing procedures and specimen management practices.

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Mutations in 16 targetable oncogenic genes were examined using reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing in 285 Chinese cervical cancers. Their clinicopathological relevance and prognostic significance was assessed. Ninety-two nonsynonymous somatic mutations were identified in 29.8% of the cancers. The mutation rates were as follows: PIK3CA (12.3%), KRAS (5.3%), HER2 (4.2%), FGFR3-TACC3 fusions (3.9%), PTEN (2.8%), FGFR2 (1.8%), FGFR3 (0.7%), NRAS (0.7%), HRAS (0.4%) and EGFR (0.4%). No mutations were detected in AKT1 or BRAF, and the fusions FGFR1-TACC1, EML4-ALK, CCDC6-RET and KIF5B-RET were not found in any of the cancers. RTK and RAS mutations were more common in non-squamous carcinomas than in squamous carcinomas (P=0.043 and P=0.042, respectively). RAS mutations were more common in young patients (<45 years) (13.7% vs. 7.7%, P=0.027). RTK mutations tended to be more common in young patients, whereas PIK3CA/PTEN/AKT mutations tended to be more common in old patients. RAS mutations were significantly associated with disease relapse. To our knowledge, this is the first comprehensive analysis of major targetable oncogenic mutations in a large cohort of cervical cancer cases. Our data reveal that a considerable proportion of patients with cervical cancers harbor known druggable mutations and might benefit from targeted therapy.

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OBJECTIVES: The objective of this study is to develop a multiplex PCR and primer extension to detect K-, N-, H-RAS, and BRAF mutations. DESIGN AND METHODS: DNA samples were isolated from 76 thyroid cancer patients. Multiplex amplification of exons 2 and 3 of three RAS genes and exon 15 of the BRAF gene using three pairs of primers was performed in a single tube. The products were split into three tubes. First, we used nine different-sized N-RAS and BRAF primers to detect base changes in N-RAS and BRAF. The other two tubes used seven separate different-sized K-RAS and H-RAS primers to detect base changes. RESULTS: We compared these results with direct sequencing. The two methods generated identical results, but our method was superior to direct sequencing in terms of the amount of work and time involved. CONCLUSIONS: We present a rapid method to detect mutations of K-, N-, H-RAS, and BRAF in human cancers.

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