Antineoplastic effect of compounds C14 and P8 on TNBC and radioresistant TNBC cells by stabilizing the K-Ras4BG13D/PDE6δ complex.

Introduction: Breast cancer (BC) is the leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) representing one of the most aggressive and treatment-resistant subtypes. In this study, we aimed to evaluate the antitumor potential of C14 and P8 molecules in both TNBC and radioresistant TNBC cells. These compounds were chosen for their ability to stabilize the complex formed by the overactivated form of K-Ras4BG13D and its membrane transporter (PDE6δ). Methods: The antitumor potential of C14 and P8 was assessed using TNBC cell lines, MDA-MB-231, and the radioresistant derivative MDA-MB-231RR, both carrying the K-Ras4B> G13D mutation. We investigated the compounds' effects on K-Ras signaling pathways, cell viability, and tumor growth in vivo. Results: Western blotting analysis determined the negative impact of C14 and P8 on the activation of mutant K-Ras signaling pathways in MDA-MB-231 and MDA-MB-231RR cells. Proliferation assays demonstrated their efficacy as cytotoxic agents against K-RasG13D mutant cancer cells and in inducing apoptosis. Clonogenic assays proven their ability to inhibit TNBC and radioresistant TNBC cell clonogenicity. In In vivo studies, C14 and P8 inhibited tumor growth and reduced proliferation, angiogenesis, and cell cycle progression markers. Discussion: These findings suggest that C14 and P8 could serve as promising adjuvant treatments for TNBC, particularly for non-responders to standard therapies. By targeting overactivated K-Ras and its membrane transporter, these compounds offer potential therapeutic benefits against TNBC, including its radioresistant form. Further research and clinical trials are warranted to validate their efficacy and safety as novel TNBC treatments.

Somatic mutation detection and KRAS amplification in testicular germ cell tumors.

Background: Testicular Germ Cell Tumors (TGCT) are the most common cancer among young adult men. The TGCT histopathology is diverse, and the frequency of genomic alterations, along with their prognostic role, remains largely unexplored. Herein, we evaluate the mutation profile of a 15-driver gene panel and copy number variation of KRAS in a large series of TGCT from a single reference cancer center. Materials and methods: A cohort of 97 patients with TGCT, diagnosed at the Barretos Cancer Hospital, was evaluated. Real-time PCR was used to assess copy number variation (CNV) of the KRAS gene in 51 cases, and the mutation analysis was performed using the TruSight Tumor 15 (Illumina) panel (TST15) in 65 patients. Univariate analysis was used to compare sample categories in relation to mutational frequencies. Survival analysis was conducted by the Kaplan-Meier method and log-rank test. Results: KRAS copy number gain was a very frequent event (80.4%) in TGCT and presented a worse prognosis compared with the group with no KRAS copy gain (10y-OS, 90% vs. 81.5%, p = 0.048). Among the 65 TGCT cases, different variants were identified in 11 of 15 genes of the panel, and the TP53 gene was the most recurrently mutated driver gene (27.7%). Variants were also detected in genes such as KIT, KRAS, PDGFRA, EGFR, BRAF, RET, NRAS, PIK3CA, MET, and ERBB2, with some of them potentially targetable. Conclusion: Although larger studies incorporating collaborative networks may shed the light on the molecular landscape of TGCT, our findings unveal the potential of actionable variants in clinical management for applying targeted therapies.

The Use of Liquid Biopsy in the Molecular Analysis of Plasma Compared to the Tumour Tissue from a Patient with Brain Metastasis: A Case Report.

Different cancers have multiple genetic mutations, which vary depending on the affected tumour tissue. Small biopsies may not always represent all the genetic landscape of the tumour. To improve the chances of identifying mutations at different disease stages (early, during the disease course, and refractory stage), liquid biopsies offer an advantage to traditional tissue biopsy. In addition, it is possible to detect mutations related to metastatic events depending on the cancer types analysed as will be discussed in this case report, which describes a patient with brain metastasis and lung cancer that harboured K-RAS mutations both in the brain tumour and in the ctDNA present in the bloodstream.

131I-C19 Iodide Radioisotope and Synthetic I-C19 Compounds as K-Ras4B-PDE6δ Inhibitors: A Novel Approach against Colorectal Cancer-Biological Characterization, Biokinetics and Dosimetry.

In 40-50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B-PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B-PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and 131I-C19 compounds as inhibitors of the K-Ras4B-PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B-PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and 131I-C19 and the tumor radiation dose were also estimated. The K-Ras4B-PDE6δ stabilizer, 131I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that 131I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.

K-RAS4A: Lead or Supporting Role in Cancer Biology?

The RAS oncogene is one of the most frequently mutated genes in human cancer, with K-RAS having a leading role in tumorigenesis. K-RAS undergoes alternative splicing, and as a result its transcript generates two gene products K-RAS4A and K-RAS4B, which are affected by the same oncogenic mutations, are highly homologous, and are expressed in a variety of human tissues at different levels. In addition, both isoforms localise to the plasma membrane by distinct targeting motifs. While some evidence suggests nonredundant functions for both splice variants, most work to date has focused on K-RAS4B, or even just K-RAS (i.e., without differentiating between the splice variants). This review aims to address the most relevant evidence published regarding K-RAS4A and to discuss if this "minor" isoform could also play a leading role in cancer, concluding that a significant body of evidence supports a leading role rather than a supporting (or secondary) role for K-RAS4A in cancer biology.

Molecules linked to Ras signaling as therapeutic targets in cardiac pathologies.

The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure.

Identification of mutant K-RAS in pituitary macroadenoma.

PURPOSE: RAS genes are among the most frequently mutated genes in cancer, where their mutation frequency varies according to the distinct RAS isoforms and tumour types. Despite occurring more prevalent in malignant tumours, RAS mutations were also observed in few benign tumours. Pituitary adenomas are examples of benign tumours which vary in size and aggressiveness. The present study was performed to investigate, via liquid biopsy and tissue analysis, the presence of K-RAS mutations in a pituitary macroadenoma. METHODS: Molecular analysis was performed to investigate K-RAS mutations using the droplet digital PCR (ddPCR) method by evaluating both plasma (liquid biopsy) and the solid tumour of a patient diagnosed with a giant clinically non-functioning pituitary tumour. RESULTS: The patient underwent surgical resection due to visual loss, and the histopathological analysis showed a gonadotrophic pituitary macroadenoma. The molecular analysis revealed the presence of mutant K-RAS both in the plasma and in the tumour tissue which, to our knowledge, has not been previously reported in the literature. CONCLUSION: Our findings highlight the exceptional capacity of the digital PCR in detecting low frequency mutations (below 1%), since we detected, for the first time, K-RAS mutations in pituitary macroadenoma. The potential impact of K-RAS mutations in these tumours should be further investigated.

Molecules linked to Ras signaling as therapeutic targets in cardiac pathologies

The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Highlights - The Ras (Rat Sarcoma) gene family is a group of small G proteins - Ras is regulated by growth factors and neurohormones affecting cardiomyocyte growth and hypertrophy - Ras directly affects cardiomyocyte physiological and pathological hypertrophy - Genetic alterations of Ras and its pathways result in various cardiac phenotypes? - Ras and its pathway are differentially regulated in acquired heart disease - Ras modulation is a promising therapeutic target in various cardiac conditions.

High-resolution melting for detecting KRAS mutations in colorectal cancer.

Colorectal cancer is a leading cause of cancer that may metastasize. KRAS gene sequence of exon 2 should be examined for identification of patients that can be treated with anti-EGFR. The aim of the present study was to evaluate the efficacy of high-resolution melting (HRM) to detect KRAS mutations in colorectal cancer (CRC) tumors. The exon 2 of KRAS was amplified from 47 adenocarcinoma CRC tissues. The tumors were subjected to high-resolution melt using quantitative PCR to identify wild-type and mutant subgroups. The results were compared to the mutations detected by next-generation sequences (NGS). The study included 47 patients, with a mean age of 62 years, of whom 24 patients were male. Most of the patients had stage II or stage III tumors. The mean melting temperatures for the wild-type and mutated group at exon 2 were 78.13˚C and 77.87˚C, respectively (P<0.001, 95% CI = 0.11-0.4). The sensitivity and specificity of high-resolution melting were 83.3 and 96.6%, respectively, with a high concordance between the NGS and HRM methods for detecting KRAS mutation in exon 2 (ĸ = 0.816; P=0.625). Thus, HRM could be used as an alternative method for detecting KRAS mutations in colorectal cancer tissue.

Current Approaches in NSCLC Targeting K-RAS and EGFR.

The research and treatment of non-small cell lung cancer (NSCLC) have achieved some important advances in recent years. Nonetheless, the overall survival rates for NSCLC remain low, indicating the importance to effectively develop new therapies and improve current approaches. The understanding of the function of different biomarkers involved in NSCLC progression, survival and response to therapy are important for the development of early detection tools and treatment options. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (K-RAS) are two of the main significant biomarkers for the management of NSCLC. Mutations in these genes were associated with development and response to therapies. For example, the use of small molecule tyrosine kinase (TK) inhibitors and immunotherapy has led to benefits in some, but not all patients with altered EGFR. In contrast, there is still no effective approved drug to act upon patients harbouring K-RAS mutations. In addition, K-RAS mutations have been associated with lack of activity of TK inhibitors. However, promising approaches aimed to inhibit mutant K-RAS are currently under study. Therefore, this review will discuss these approaches and also EGFR therapies, and hopefully, it will draw attention to the need of continued research in the field in order to improve the outcomes in NSCLC patients.

Cáncer Colorectal: características de la expresión del oncogen K-ras / Colorectal cancer: characteristics of the expression of K-ras oncogene

Introducción: El cáncer de colon ocupa el cuarto lugar como causa de muerte por cáncer en adultos. Objetivo: Describir características de la expresión del oncogen K-ras en pacientes con cáncer colorectal (CCR) que acudieron a consulta de Gastroenterología del Hospital Universitario de Caracas en el período enero-julio 2014. Metodología: Estudio de corte transversal, descriptivo y prospectivo. La población de estudio estuvo conformada por pacientes con diagnóstico de CCR por colonoscopia e histología con evaluación molecular del K-ras. Resultados: de 35 pacientes 57,14% fueron del sexo masculino y 42,86% del femenino con edad media de 57±17años; el 100% de la muestra por histología correspondió a ADC predominando el tipo moderadamente diferenciado (40,00%). 28 pacientes (80%) no presentó mutación del K-ras mientras que 7 (20,00%) sí, de los cuales 6 (85,71%) reportó mutación en el codón 12 y 1 en el 13 (14,28%); en 4 de los 7 pacientes (57,14%) la mutación estuvo en el colon izquierdo. Conclusiones: la mutación del K-ras predomina en el sexo masculino con edad media de 57años estando presente en 20% de la población; la mutación en el codón 12 es más frecuente asociada al colon izquierdo y el CCR más común es el ADC bien diferenciado.

Introduction: Colon cancer ranks fourth leading cause of cancer death in adults. Objective: To describe characteristics of the expression of K-ras oncogene in patients with colorectal cancer (CRC) who attended the outpatient Gastroenterology of the University Hospital of Caracas (HUC) in the period from january-july 2014. Methodology: Transversal, descriptive and prospective court. The study population consisted of patients diagnosed with CRC by colonoscopy and histology with molecular evaluation of K-ras. Results: Of the 35 patients 57,14% were male and 42,86% female with a mean age of 57±17 years old; 100% of the sample corresponded to ADC histology predominating moderately differentiated rate (40.00%). 28 patients (80%) had no mutation of K-ras while 7 (20,00%) did, of which 6 (85,71%) reported mutation at codon 12 and 1 in the 13 (14,28%); in 4 of 7 patients (57,14%) mutation was in the left colon. Conclusions: K-ras mutation predominates in males with an average age of 57 years old and is present in 20% of the population; mutation in codon 12 is most frequently associated with the left colon and the most commom type of CRC by histology is the well differentiated ADC.

[Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population]. / Relevancia clínica del oncogén K-ras en cáncer de colon, experiencia en una población mexicana.

BACKGROUND: Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. AIMS: To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. METHODS: Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. RESULTS: No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). CONCLUSIONS: No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis.

Arginine vasopressin controls p27Kip1 protein expression by PKC activation andirreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Rasdependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated â-galactosidase (SA-âGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Rasdependent Y1 malignant cells by AVP requires large quantities of the p27Kip1 protein as well as phosphorylation of the p27Kip1 protein at both Ser10 and Thr187.

Frecuencia y asociación línicopatológico de las mutaciones del oncogen K-ras en pacientes venezolanos con cáncer colorectal / Frequency and clinicopathological associations of K-ras mutations in Venezuelan patients with colo-rectal cancer

Las mutaciones en el oncogén K-ras son comunes en cáncer colo-rectal, afectan el comportamiento biológico y podrían influir en la susceptibilidad terapéutica en estos tumores. El objetivo de este trabajo fue identificar los tipos de mutación K-ras observados en pacientes referidos con cáncer colo-rectal y relacionarlos con el grado de diferenciación histológica y con el estadio clínico. Se obtuvo ADN genómico tanto de tejido tumoral incluido en parafina, como de tejido fresco. Se amplificó el gen K-ras a través de la reacción en cadena de la polimerasa (RCP) y se digirieron los fragmentos amplificados con enzimas de restricción, por último se obtuvieron datos clínicos e histopatológicos de las historias clínicas. Se encontraron mutaciones en los codones 12 y 13 del oncogén K-ras en el 23,33% de los pacientes. De estos 28,57% en el codón 12, en el codón 13 se encontró un 57,14% y 14,29% para ambos codones. Fueron más frecuentes en el hemicolon izquierdo con 78,57% y según la clasificación histológica en los adenocarcinomas bien diferenciados (58,70%) y en los mucinosos (28,57%). Las mutaciones identificadas fueron mas frecuentes en estadios avanzados C2 de la clasificación de Dukes`. El análisis molecular del oncogén K-ras permitió evidenciar mutaciones que sirven como parámetro diagnóstico y pronóstico en los tumores colo-rectales. La frecuencia de mutaciones encontradas en este trabajo es similar a algunas de las reportadas a nivel mundial, sin embargo difieren en el tipo de mutación mas frecuente, que en nuestro medio fue la mutación del codón 13 del gen con más de un 50%.

Mutations in the K-ras oncogene are common in colo-rectal cancer, which affect the biological behaviour and may influence the susceptibility to therapy in these tumors. The objective of this work was to identify the types of K-ras mutations observed in referred patients with colo-rectal cancer and to relate them to their degree of histological differentiation and clinical stage. Histopathological and clinical data were obtained from medical records. DNA was obtained from both, fresh tissue and tumor tissue embedded in paraffin. The K-ras gene was amplified through the polymerase chain reaction (PCR) and the amplified fragments were digested with restriction enzymes. We found mutations in codons 12 and 13 of the K-ras oncogene in 23.33% of patients. Of these, 28.57% were located at codon 12, 57.14% were at codon 13 and 14.29% at both codons. They were more frequent in tumors located in the left hemicolon and, according to their histological type, were more frequent in well differentiated adenocarcinomas (58.70%) and in mucinous (28.57%). The identified mutations were more frequent in advanced stages (C2) of Dukes’ classification. The molecular analysis of the K-ras oncogene made mutations evident, which could be useful in the diagnosis and prognosis of colorectal tumors. The frequency of mutations found in this work is similar to some of those reported worldwide; however, they differ in the more frequent type of mutation, which, in our study, was located at codon 13 in more than 50% of the cases.

Detección de mutaciones en los genes K-ras, H-ras y EGFR en muestras de plasma sanguíneo y cepillado cervical de pacientes con neoplasia intraepitelial cervical (NIC) III y cáncer de cuello uterino / Detection of genes mutations in the K-RAS, H-RAS and EGFR in samples of blood plasma and cervical smears for patients with cervical intraepithelial neoplasia III and cervical cancer

Introducción: El cáncer cervical es el segundo cáncer más importante en mujeres a nivel mundial y la segunda causa de muerte en mujeres por cáncer. Se ha demostrado que el proceso de carcinogénesis cervical presenta componentes tanto genéticos, epigenéticos y medio ambientales. En la actualidad, muchos estudios se encaminan en la búsqueda de marcadores moleculares como mutaciones en oncogenes y/o genes tumor supresor que se asocien con la progresión de esta entidad. Los genes candidatos más estudiados en cáncer cervical en distintas poblaciones han sido H-ras, K-ras, EGFR entre otros. Objetivos: Se identificó el virus de papiloma humano (VPH) genérico y específico en el ADN libre de plasma y de cepillado cervical de pacientes con cáncer cervical invasivo y con neoplasia intraepitelial cervical (NIC) III además de evaluar alteraciones genéticas, como mutaciones en los genes H-ras, K-ras y EGFR. Metodología: Para ello se detectó el VPH genérico mediante PCR con los iniciadores GP5+/GP6+, y específico para VPH 16 y 18 en la región E6/E7. Para detectar las mutaciones en el codón 12 de H-ras, codones 12 y 13 de K-Ras y el exón 21 de EGFR se realizó mediante secuenciación directa de los productos de PCR de estos fragmentos génicos. Resultados: Obteniendo una buena correlación entre las muestras de plasma sanguíneo y los cepillados cervicales, tanto para los hallazgos de VPH p=0.0374 como para las mutaciones evaluadas p=0. En general, para EGFR en el exón 21 no se encontraron mutaciones, al igual que para los codones 12 y 13 en K-ras y codón 12 en H-ras. Conclusión: El uso del ADN presente en el plasma puede ser relevante para el análisis de mutaciones y de la presencia de marcadores tumorales cuando no se dispone de otras muestras.

Introduction: Cervical cancer is the second most important cancer in women worldwide, and the second cause of cancer death in women. It has been shown that the process of cervical carcinogenesis presents as genetic and epigenetic components as environmental issues. At present, many studies are addressed in searching for molecular markers such as mutations in oncogenes and/or tumor suppressor genes that are associated with the progression of this disease, the most studied candidate genes in cervical cancer in different populations have been H-ras, K-ras, EGFR among others. Objective: The present study identified human papilloma virus (HPV) generic and specific in DNA-free plasma and cervical smears of invasive cervical cancer patients and patients with cervical intraepithelial neoplasia (CIN) III in addition to assessing genetic alterations, such as mutations in the genes H-ras, EGFR and K-ras. Methods: To do so generic HPV was detected by PCR with primers GP5+/GP6+, and specific HPV 16 and 18 in E6/E7 region; to detect mutations in codon 12 of  H-ras, codons 12 and 13 of K-ras and EGFR exon 21 was conducted by direct sequencing of PCR products of these gene fragments. Results: Getting a good correlation between samples of blood plasma and cervical smears for both; the findings of HPV p=0.0374 and evaluated mutations p=0. In general, for EGFR in exon 21 mutations were not found, as for codons 12 and 13 in K-ras and codon 12 in H-ras. Conclusion: The use of DNA in plasma may be relevant to the analysis of mutations and the presences of tumor markers are not available from other samples.