Point Mutations Of Ras Oncogenes Research Articles

Conformational SERS Classification of K-Ras Point Mutations for Cancer Diagnostics.

Point mutations in Ras oncogenes are routinely screened for diagnostics and treatment of tumors (especially in colorectal cancer). Here, we develop an optical approach based on direct SERS coupled with chemometrics for the study of the specific conformations that single-point mutations impose on a relatively large fragment of the K-Ras gene (141 nucleobases). Results obtained offer the unambiguous classification of different mutations providing a potentially useful insight for diagnostics and treatment of cancer in a sensitive, fast, direct and inexpensive manner.

Studies on the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer.

To study the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer. The point mutations at codon 12 and 61 of c-Ha-ras, at codon 12 and 13 of K-ras, and at codon 12 of N-ras were studied with PCR-RFLP in 88 formalin fixed and paraffin embedded specimens of gastric cancer. It was found that the overall rate of point mutation of ras oncogenes was 18.2% and the positivity of the point mutation of ras oncogenes was related to the cancerous invasion of the serosa, the status of lymph node metastasis, the stage of cancer and the survival time after surgery. The findings suggest that the determination of point mutations of ras oncogenes can be used to determine the prognosis of patients with gastric cancer.

Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

Prevalence of RAS Point Mutations in Papillary Thyroid Carcinoma; A Novel Mutation at Codon 31 of K-RAS

The aim of this study was to assess the incidence of point mutations in RAS oncogenes of papillary thyroid carcinoma (PTC). Tumour specimens were obtained from 29 PTCs. The fragments of exons 1 and 2 of RAS oncogenes family (H- RAS, K- RAS, N- RAS) were amplified and then, point mutations were detected by SSCP and/or by RFLP analysis. Several DNA samples were directly sequenced to confirm the results. Two mutations were found in this study (GAA/CAA at codon 31 of K- RAS and CAA/CAC at codon 61 of N- RAS oncogene). These data confirm the results of previous studies, showing that RAS mutations are more rarely found in PTC than in follicular neoplasms. The influence of a novel mutation at codon 31 of K- RAS oncogene on the development of PTC needs further studies.

Farnesyl transferase inhibitors: the next targeted therapies for breast cancer?

The ras family of proto-oncogenes are upstream mediators of several essential cellular signal transduction pathways involved in cell proliferation and survival. Point mutations of ras oncogenes result in constitutively active Ras and have been shown to be oncogenic. However, ras activation can occur in the absence of ras mutations secondary to upstream receptor activation. The first important step in Ras activation is farnesylation by farnesyl transferase, and inhibitors of this enzyme have been demonstrated to inhibit Ras signaling, and have anti-tumor effects. However, it is now clear that farnesyl transferase inhibitors (FTIs) have activity independent of Ras, most likely due to effects on prenylated proteins downstream of Ras, which explains their activity in several malignancies, including breast cancer, where ras mutations are rare. Several FTIs are in clinical development for the treatment of solid tumors. Preclinical evidence suggests that FTIs can inhibit breast cancers in vitro and in vivo, and a phase II trial of the FTI, R115777, in patients with advanced breast cancer produced encouraging results. Based on prior successful outcomes with agents targeting the estrogen and epidermal growth factor receptor pathways in breast cancer, the FTIs, used alone or more likely with other agents, may be the next exciting targeted therapy in breast cancer.

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Exploring environmental causes of altered ras effects: fragmentation plus integration?

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects.

Detection of Ras Oncogene Point Mutations and Simultaneous Proliferative Fraction Estimation in Gallbladder Cancer

Gallbladder cancer is notorious for its poor clinical evolution; so, a study of parameters with any prognostic potential is of particular interest. In this study, we investigated 23 adenocarcinomas regarding both the presence of point mutations in the ras gene family and the quantitative expression of Proliferating Cell Nuclear Antigen (PCNA). We respectively used molecular biology techniques and immunohistochemistry. Our results were related to several clinicopathologic determinators as well as to patients' survival. Mutations in codon 12 of the K-ras gene were detected in four gallbladder neoplasms (17%). This specific type of mutation is likely to be partially involved in this organ's tumourigenesis, particularly since no H-ras codon 12 or K-ras codon 13 (aspartic acid) mutations were detected in any of our specimens. PCNA immunoreactivity was generally limited in all tumours studied except for those morphologically characterized as being particularly aggressive. Notably, the four tumours in which K-ras mutation was detected, demonstrated relatively high PCNA positive immunoexpression in their malignant cells. This finding reinforces a possible correlation between the presence of ras mutations and increased cell growth. Staging was the only factor which was statistically associated with survival (p < 0.01). Therefore, the poor evolution of this cancer is probably due to late diagnosis and not related to a model of increased biologic aggressiveness.

Radiolabeled polymerase chain reaction assay for detection of ras oncogene point mutations in tumors

The human ras gene plays a fundamental role in the transduction of extracellular signals to the nucleus, thereby regulating cell growth and differentiation. Point mutations in the ras gene convert it into a transforming oncogene that has been found in many solid and hematologic malignancies. We describe a rapid and sensitive assay based on a radiolabeled polymerase chain reaction followed by restriction enzyme digestion that we have adapted for differentiating between the wild-type and mutant ras genes. This assay should prove useful in the analysis of ras gene point mutations in clinical tumor specimens in which ras oncogene activation is an early event in carcinogenesis.

The rat N-ras gene; interference of pseudogenes with the detection of activating point mutations.

The PCR technique in combination with selective hybridization to mutation specific oligonucleotides, is a widely used methodology for the detection of activating point mutations in ras oncogenes. In the present paper we demonstrate for the N-ras gene of the rat that processed pseudogenes do interfere with this method. A first indication for this interference came from the sequence analysis of cloned PCR fragments of exon 1, amplified with primers derived from previously reported exon sequences of the mouse N-ras gene. Between different clones originating from one PCR reaction, a marked sequence heterogeneity is observed and this is shown to be the result of the presence of at least two different processed pseudogenes of the rat N-ras gene. These two pseudogenes, together with the wildtype N-ras gene and a small 3' part of the unr gene, were eventually cloned and their genomic organization and nucleotide sequences determined. Furthermore, representative examples of the confounding effects of these pseudogenes on the screening for activating point mutations are presented. Taken together, our results demonstrate that intron-specific amplification is a prerequisite for the unambiguous detection of activating point mutations in the N-ras gene of the rat.

RARE FREQUENCEY OF POINT MUTATIONS FOR CODON 12, 13 AND 61 OF RAS GENE IN ITALIAN NEUROBLASTOMA

Single point mutations of ras oncogenes are found in many tumors and contribute to the pathogenesis of the cancer. The product of the ras gene, p21 protein, was found expressed in several neuroblastoma tissues. However, the role of ras gene in this tumor has yet to be clarified. To contribute to the understanding of the ras activation, 79 fresh biopsies of neuroblastoma were studied to investigate the possibility that ras would be activated by point mutation. Analysis of H-ras and N-ras was performed by means of PCR and SSO, while K-ras mutations were detected by multiplex-ASPCR. None of the neuroblastomas examined showed H- or K-ras activation, while N-ras mutations were demonstrated in only three patients (3,7%). N-myc oncogene is amplified in a substantial number of patients with neuroblastoma. N-myc amplification was studied by Southern blot technique. N-myc amplification was demonstrated in 13.2% of patients less than 1 year of age at diagnosis and 23% of older children. Two of the patients (one stage I and one stage IVs) with N-ras mutation and without N-myc amplification had a good outcome, while the third (stage IVs) with N-myc amplification had a poor prognosis. These results suggest that ras activation is a rare event in both amplified and non-amplified neuroblastoma tumors and that N-ras activation was not involved in the clinical outcome of these patients. Moreover, our data suggest that p21 expression is induced by a post-transcriptional activation.

Detection of High Incidence of H‐RAS Oncogene Point Mutations in Acute Myelogenous Leukemia

We have been analyzing RAS p21 proteins and the DNA sequence of leukemic cells. We report here that these cells have high expression of H-RAS p21, which originates from point mutations of RAS oncogenes. The leukemic cells from six patients with acute myelogenous leukemia were separated from heparinized whole blood and bone marrow by a density gradient technique. The expression of RAS oncogenes was analyzed by a fluorescence-activated cell sorting with a panel of monoclonal antibodies. The high expression of DWP, which was reported to recognized activated RAS oncogene, was found in two patients and was associated with high levels of H-RAS expression. These facts prompted us to analyze the DNA sequence of RAS genes with an automated DNA sequencer. Unexpectedly, various kinds of H-RAS point mutations were found in all six cases, including two cases of hot-spot point mutation at codon 12, whereas K-RAS point mutation (no hot-spot point mutations) was found in six cases. The same H-RAS point mutations, at codons 10, 11, and 15, were found in all six cases. To our knowledge, there is no report on H-RAS point mutation in human leukemias. On the basis of these findings, we suggest that H-RAS point mutation together with p53 gene mutation may play an important role in leukemogenesis.

Clinical significance of ras oncogene activation in human lung cancer : Rodenhuis S, Slebos RJC. Division of Experimental Therapy, Department of Medical Oncology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam. Cancer Res 1992;52:Suppl. 2665s-9s

Activation of ras oncogenes is commonly found in human neoplasms. We have investigated 280 human lung cancer specimens for ras activation, including 38 that have not been reported previously, using an oligonucleotide detection assay. From a total of 141 adenocarcinoma samples from smokers, 41 tested positive for a point mutation in codon 12 of K-ras (30%), while three tumors had another type of ras activation. Only two of 40 cases from nonsmokers had a K-ras mutation (5%), suggesting that K-ras mutations may be directly caused by exposure to carcinogens in tobacco smoke. The majority of the point mutations in adenocarcinomas were guanine to thymine transversions in codon 12 of the K-ras oncogene. Occasional point mutations in ras oncogenes were detected in adenosquamous carcinomas (one of five cases) and large cell carcinoma (one of 24 cases), but no ras activations were found in small cell carcinomas (six cases), squamous carcinomas (48 cases), carcinoid carcinomas (15 cases), or thymoma (one case). Analysis of the clinical and pathological features of the adenocarcinoma cases showed no apparent associations between the K-ras activation and age at diagnosis, sex, disease stage, and the occurrence of other neoplasms. K-ras-positive adenocarcinomas tended to be less differentiated than the K-ras-negative ones (P = 0.044, chi 2 test for trend). K-ras mutations identify a subgroup of patients with adenocarcinoma of the lung who have a very poor prognosis despite radical resection of their tumor. Although K-ras has been proposed as a target for antitumor therapy, its major clinical significance could be to aid in the selection of patients for specific therapeutic interventions, such as adjuvant chemotherapy.

Solitary Polyclonal Autonomous Thyroid Nodule: A Rare Cause of Childhood Hyperthyroidism*

Solitary autonomous thyroid tumors are an unusual cause of hyperthyroidism, particularly in childhood. We describe the youngest individual so far reported with this condition, a 22 month child with a large hyperfunctioning thyroid nodule who became overtly hyperthyroid after iodinated contrast administration. The histology of the nodule was compatible with follicular cell hyperplasia. These tumors are often called toxic adenomas, although there is no solid evidence that they are true neoplasms. We examined the clonal composition of the child's thyroid tumor by X-chromosome inactivation analysis, taking advantage of a polymorphism in the X-chromosome gene phosphoglycerate kinase. The tumor consisted of an even mixture of cells containing activated paternal and maternal PGK alleles, indicating that the tumor was polyclonal. Furthermore, the nodule had no structural rearrangements or activating point mutations of ras oncogenes, which are found in up to 50% of solitary monoclonal follicular adenomas. Solitary hot nodules may at least in some cases be secondary to hyperplasia, and not to clonal expansion of an abnormal, mutated cell. This may also explain the relatively low frequency of malignant transformation observed in hyperfunctioning thyroid tumors.

Point Mutations of Ras Oncogenes are an Early Event in Thyroid Tumorigenesis

Identifying the nature of the genetic mutations in thyroid neoplasms and their prevalence in the various tumor phenotypes is critical to understanding their pathogenesis. Mutational activation of ras oncogenes in human tumors occurs predominantly through point mutations in two functional regions of the molecules, codons 12, 13 (GTP-binding domain) or codon 61 (GTPase domain). We examined the prevalence of point mutations in codons 12, 13, and 61 of the oncogenes K-ras, N-ras, and H-ras in benign and malignant human thyroid tumors by hybridization of PCR-amplified tumor DNA with synthetic oligodeoxynucleotide probes. None of the eight normal thyroid tissues harbored point mutations. Four of nineteen nodules from multinodular goiters (21%), 6/24 microfollicular adenomas (25%), 3/14 papillary carcinomas (21%), and 0/3 follicular carcinomas contained ras point mutations. The predominant mutation was a valine for glycine substitution in codon 12 of H-ras. None of the multinodular goiter tumors known to be polyclonal (and thus due to hyperplasia) had point mutations, whereas one of the two monoclonal adenomas arising in nodular glands contained in H-ras codon 12 valine substitution, which was confirmed by sequencing the tumor DNA. These data show that ras activation is about equally prevalent in benign and malignant thyroid neoplasms, and thus may be an early event in the tumorigenic process.

RAS Mutations Are Rare Events in Philadelphia Chromosome-Negative/bcr Gene Rearrangement-Negative Chronic Myelogenous Leukemia, But Are Prevalent in Chronic Myelomonocytic Leukemia

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

RAS mutations are rare events in Philadelphia chromosome-negative/bcr gene rearrangement-negative chronic myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome- positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

Infrequent point mutations of ras oncogenes in gastric cancers

Adenocarcinomas of the proximal and distal stomach have significant clinical and biological differences. A study was undertaken to determine if a difference in the incidence of mutated ras oncogenes exists between proximal (gastroesophageal junction or cardia) and distal (body or antrum) gastric tumors, and to assess the overall incidence in gastric cancers from non-Asian patients. Deoxyribonucleic acid from 28 primary gastric adenocarcinomas were analyzed for point mutations in codons 12, 13, and 61 of the Ha- ras, Ki- ras, and N- ras protooncogenes using polymerase-catalyzed chain reaction methodology. Twelve tumors were located at the gastroesophageal junction or cardia, and 16 in the body or antrum. Mutated ras genes were detected in 2 of 28 tumors for an overall incidence of 7%. The mutations occurred in codon 61 of the N- ras gene in a proximal gastric cancer and in codon 12 of the Ki- ras gene in a distal gastric cancer. These data indicate that mutations in ras genes are an uncommon event in primary gastric cancers and that there is no meaningful difference in the incidence of ras mutations in proximal and distal stomach cancers.