Single Genetic Alteration Research Articles

Distinguishing de Novo Second Cancer Formation from Tumor Recurrence : Mutational Fingerprinting by Microdissection Genotyping

Patients with one form of cancer are known to possess a higher risk for development of a second tumor, presenting synchronously or metachronously over time. Distinguishing whether the second tumor represents a de novo cancer or a recurrence/metastasis of the first cancer has important implications for treatment and prognosis and specific clinical and pathological evidence is sought to resolve this issue. De novo cancer formation is favored by long latency interval (usually exceeding five years), better differentiation in the subsequent tumor, solitary second tumor formation and occurrence of the later tumor in a site not typical of metastatic spread. Conversely, recurrent metastatic disease is favored by short interval to second cancer formation, similar histology with increased anaplasia in the second tumor and lack of in situ malignancy and multifocal tumor deposits. While these criteria are sufficient in most instances to distinguish between the two, varying degrees of uncertainty can persist with a minority of cases remaining unresolved.

Loss of p16 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation.

Ewings sarcoma and primitive neuroectodermal tumors (ES/PNET) are characterized by the fusion of the N-terminus of the EWS gene to the C-terminus of a member of the ETS family of transcription factors. While such fusion proteins are thought to play dominant oncogenic roles, it is unlikely that a single genetic alteration by itself will support cellular transformation. Given that EWS/FLI1 is only able to transform immortalized 3T3 fibroblasts and that 30% of ES/PNET tumors contain a homozygous deletion of the p16 locus, it is likely that other genetic events are required for EWS/FLI1 oncogenesis. Here we describe a complementary mechanism utilized in the establishment ES/PNET tumors. EWS/FLI1 has the capacity to induce apoptosis and growth arrest in normal MEFs. Such effects prevent the establishment of stable expression of the protein in these cells. When expressed in p16, p19(ARF), or p53 deficient MEFs, the apoptotic and growth arrest effects are attenuated, creating a environment permissive for stable expression of the protein. While loss of a single tumor suppressor is sufficient to establish expression of EWS/FLI1, cellular transformation requires further genetic perturbation.

Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis

To evaluate how characterization of genetic alterations can help in the elucidation of liver carcinogenesis pathways, 137 tumors were analyzed. High-density allelotype, p53, Axin1, and beta-catenin gene mutations were determined. Alterations were analyzed according to clinical parameters. Tumors could be divided into 2 groups according to chromosome stability status. In the first group, demonstrating a chromosome stability, beta-catenin mutation associated with chromosome 8p losses were frequently found as the single genetic alterations. beta-catenin mutations were associated with large tumor size and with negative hepatitis B virus status. In the second group, demonstrating a chromosome instability, the most frequent allelic losses were on chromosome 1p, 4q, 6q, 9p, 13q, 16p, 16q, and 17p; Axin1 and p53 were frequently mutated. All of these alterations, except losses on 6q and 9p, were associated with hepatitis B virus infection. P53 mutations, 17p, 13q losses, and a high value of the fractional allelic loss index were associated with poor differentiated tumors, independently of risk factors. Finally, in the whole series, chromosome 9p and 6q losses were associated with poor prognosis. Two main pathways defined by genetic alterations show different risk factors and clinical characteristics. Furthermore, loss of chromosome 9p or 6q is an independent prognostic indicator.

Pilocytic astrocytomas do not show most of the genetic changes commonly seen in diffuse astrocytomas.

While it is well known that pilocytic astrocytomas are clinically distinct from diffuse astrocytomas, few comprehensive studies have focused on their genetic differences. The aim of this study was to examine pilocytic astrocytomas for genetic alterations that are commonly seen in diffuse astrocytomas. By using molecular genetic and immunohistochemical techniques, we evaluated p16, p53, CDK4 and PTEN genes in 29 pilocytic astrocytomas. Mutation screening of p53 and PTEN was performed by single strand conformation polymorphism analysis followed by direct sequencing. Loss of heterozygosity (LOH) of p53, p16 and 10q23-25 loci was performed with microsatellite markers and genomic microsatellite instability (MSI) was also screened. Protein expression of p16, p53, CDK4 and PTEN was examined by immunohistochemistry. Five tumours were found to have single genetic alterations, which included a p53 mutation, a PTEN mutation, MSI at a single microsatellite marker of the p16 locus, and one single LOH at each p16 and 10q23 loci. Protein expressions of p16, CDK4 and PTEN were detected in 73%, 61% and 38% of tumours, respectively. Significantly and in sharp contrast to diffuse astrocytomas, no pilocytic astrocytoma in our series stained for p53 protein. Pilocytic astrocytomas have neither MSI phenotype nor recurrent alterations of the p53 and p116 genes. However, altered expression of PTEN may be important in the genesis of pilocytic astrocytomas. We conclude that pilocytic astrocytomas are genetically distinct from diffuse astrocytomas. Lack of p53 mutation/immunostaining may serve as a diagnostic adjunct for differentiating pilocytic astrocytomas from diffuse astrocytomas in small neurosurgical biopsies.

Cardiovascular News

Cardiovascular News

Variable genetic alterations and survival in head and neck cancer.

To evaluate multiple genetic loci in patients with head and neck cancer to determine if, as in colorectal carcinoma, there is an orderly occurrence of genetic alterations, and if an accumulation of alterations affects patient survival. Cohort study of patients with head and neck cancer in which fresh tissue was retrieved. Academic medical center. Forty-three patients treated surgically for squamous cell carcinoma of the head and neck from 1991 to 1994. The DNA from tumor and healthy tissue was evaluated for loss of heterozygosity at p53, retinoblastoma, and chromosome 16q and for amplification of cyclin D1. The respective RNA was probed for levels of p53, p 16, p21, and p27 messenger RNA. These findings were compared with tumor stage and patient survival. DNA analysis showed that loss of heterozygosity occurred at p53 in 21% of tumors, at retinoblastoma in 35%, and at 16q in 21%, and that cyclin D1 was amplified in 42%. Messenger RNA levels of the assessed proteins were variably increased and decreased compared with healthy tissues obtained from the same patients with no discernable pattern. There was no correlation between any one of these genetic alterations and overall survival. When patients were analyzed for loss of heterozygosity at p53, retinoblastoma, 16q, or altered cyclin D1 in combination, 19 patients had no detectable alterations, 13 had 1, 6 had 2, and 5 had 3. Single genetic alterations did not affect survival; however, there was a trend toward decreased survival with multiple alterations. The 2-year Kaplan-Meier survival in patients with less than 1 genetic loss was 78% vs 58% in patients with 2 or more losses. The lack of a pattern of genetic alterations in head and neck cancer demonstrates that its progression can be mediated by a multitude of pathways, complicating its genetic evaluation. Single genetic alterations do not appear to affect survival; however, when multiple alterations are detected-regardless of combination-survival is affected. This observation lends credence to the theory that multiple genetic alterations contribute to cancer progression; however, the lack of a pattern of this genetic change is a significant obstacle to applying genetic findings to routine cancer therapy.

Genetic and molecular basis of cardiac arrhythmias: impact on clinical management part III.

In Parts I and II of this article,* we discussed monogenic arrhythmic disorders. These are determined or favored by an inborn alteration and for the most part are characterized by a single genetic alteration. This has allowed the use of “paradigms”; namely, diseases, such as the long-QT syndrome (LQTS), in which it has been possible to trace specific mutations on ion channel genes to their electrophysiological consequences in the patient. Unfortunately for the practicing cardiologist, these “simple” diseases constitute only a small part of the clinical conditions associated with cardiac arrhythmias. The majority of cases affect patients in whom the arrhythmogenic substrate is complex. Indeed, the expression of the molecular systems responsible for normal and abnormal electrical activity vary significantly, depending on a variety of factors, including age, regional factors (type of cells, myocardial perfusion), and such underlying chronic diseases as cardiac hypertrophy, myocardial infarction, and heart failure. The study of this complex system of interacting molecular functions requires an approach somewhat different from that required to consider monogenic disease. Accordingly, in this section we discuss broader themes that are essential to understand the integration of gene expression, ion channel function, and cell coupling in multicellular networks as a first step toward the comprehension of more frequent and more complex arrhythmogenic conditions. ### Diversity of Gene Expression in the Heart Understanding cell-to-cell variability in the cardiac action potential shape and the mechanisms underlying impulse propagation is the key to understanding normal and abnormal cardiac electrophysiology. Much of this variability can be attributed to variability in the characteristics of individual ion currents whose integrated behavior determines the shape and duration of action potentials in individual cardiac cells, as well as to variability in cell-to-cell communications. Ion currents are now recognized to flow through specific pore-forming membrane proteins called ion channels. The first gene encoding an ion channel protein was …

Linkage of Infantile Bartter Syndrome with Sensorineural Deafness to Chromosome 1p

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.

The Iodine Factor in Health and Evolution

Iodine is essential for modern humans and may have been essential for Neandertals as well. Today about 30 percent of the world's population is at risk of iodine deficiency disorders (idd), 750 million people suffer from goiter, 43 million have idd‐related brain damage and mental retardation, and 5.7 million are afflicted by cretinism, the most severe form of idd. Distinctive Neandertal skeletal traits are identical to those of modern humans who suffer from cretinism. Cro‐Magnon Venus figurines also exhibit distinctive traits associated with cretinism among modern humans. This new evidence, coupled with recent mitochondrial dna findings, suggests that a single genetic alteration, which improved the ability of the thyroid gland to extract and utilize iodine, may account for differences between Neandertals and modern humans. Late Pleistocene human evolution, consequently, may be explained by several alternative interpretations involving iodine pathology and/or biological adaptation. Speciation may have resulted from the geographical isolation of inland populations.

An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.

The human lymphoblastoid MT1 B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT1 cells nevertheless remain sensitive to mutagenesis by MNNG and display a mutator phenotype. These phenotypes have been attributed to a single genetic alteration postulated to confer a defect in strand-specific mismatch repair, a proposal that attributes the cytotoxic effect of DNA alkylation in wild-type cells to futile attempts to correct mispairs that arise during replication of alkylated template strands. Our results support this view. MNNG-induced mutations in the HPRT gene of MT1 cells are almost exclusively G.C-->A.T transitions, while spontaneous mutations observed in this mutator cell line are single-nucleotide insertions, transversions, and A.T-->G.C transitions. In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight base-base mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a required activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mammalian cells.

Pit-1 and hypopituitarism

The story of Pit-1 and hypopituitarism in humans provides an excellent example of pleiotrophism or multiple phenotypic effects resulting from a single genetic alteration. It shows how defects in this single gene cause the absence o f several pituitary hormones. Three recent articles reviewed here provide examples of different mutations in this homeobox gene encoding a transcriptional activation protein that is vital to the embryologic development, survival, and differentiated function of somatotropes, lactotropes, and thyrotropes.

Somatic recombination of the mnd chromosomal region in diploids and dikaryons of Schizophyllum commune

Somatic recombination was compared in genetically identical diploid and dikaryotic mycelia heteroallelic for the recessive morphological marker, mnd, and six other genetic markers. Although recombination including the chromosomal segment containing mnd was detected in both types of somatic cells, the underlying process appears to be different. The diploids showed parasexual recombination associated with sectoring, whereas the dikaryon typically showed, with rare exception, a peculiar type of internuclear transfer resulting in a single genetic alteration in the mnd chromosomal region. Some of the haploid mnd end products recovered from diploids produced unusual and unstable dikaryotic transformed mound tissue when mated to mnd + strains. The instability produced normal dikaryotic sectors which proved upon analysis to be revertants to nonmounds. The results suggest that these “transformed” dikaryotic mound cells contained a donor mnd nucleus and a recipient nucleus which is somehow heteroallelic [mndmnd +]. Reversion from a mound to a nonmound cell is envisioned as loss of the previously acquired mnd allele. These results suggest that unstable mounds formed in dikaryotic colonies do not occur by a somatic recombination mechanism involving allelic substitution, as previously hypothesized, but rather by the addition of genetic material to the mnd + nucleus without genetic loss. Whatever the precise mechanism for stable and unstable mound development in dikaryons, the studies detailed within suggest that a diploid nucleus is an unlikely intermediate in the somatic recombination process.

Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.

Isolation and preliminary characterization of Sindbis virus-resistant Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells resistant to the cytolytic effects of Sindbis virus infection have been selected in one step from mutagenized wild-type cells. The mutants were present at frequencies as high as 8.8 × 10 −4, and were obtained virus free following the incubation of the isolates in the presence of anti-Sindbis virus antibody. The mutant phenotypes behaved recessively in somatic cell hybrids, and there were at least two complementation groups among the isolates tested. In addition to the virus-resistant phenotype, the mutants clearly have surface changes as demonstrated by their altered morphology, increased sensitivity to phytohemagglutinin, and increased resistance to diphtheria toxin. Analyses of revertants from one mutant indicated that all of the phenotypes of the mutant resulted from a single genetic alteration. All isolates were partially defective in viral adsorption. Comparison of viral replication in WT and one mutant cell strain suggested that the major effect on viral replication in the mutant studied is at the level of viral mRNA translation.

Osmotic-Sensitive Mutant of Salmonella typhimurium

A strain (DA82) having peculiar osmotic properties was isolated in Salmonella typhimurium. The mutant shows increased elasticity of its cell wall and makes spherical instead of elongated cells, regardless of the osmolality of the medium. The strain withstands dilution in distilled water without disruption or death and grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies exponentially in low-osmolality broth (40 milliosmol). Addition of salts or sucrose instantly stops death and allows growth and cell division to proceed. Death is not due to lysis because this appears at later times and at a much lower rate. Osmotic inactivation is temperature-dependent: higher death rates occur at higher incubation temperatures. Inhibition of protein synthesis by chloramphenicol (20 mug/ml) prevents osmotic death. At 37 C and at lower temperatures, the phenomenon of osmotic death is transient. After a variable interval, growth of the osmotic-sensitive strain resumes. It is assumed that the strain's osmotic behavior is due to membrane defectiveness. The membrane disfunction and the wall defect shown by the strain may be consequences of a single genetic alteration or the results of independent mutations.

Studies of a phenylalanine-tyrosine requiring mutant of Neurospora crassa (strain S 4342)

A mutant of Neurospora crassa, strain S 4342, which requires phenylalanine and tyrosine was recovered by a modified filtration method with a microconidial strain. The double nutritional requirement was found due to a single genetic alteration at a locus other than that of known mutants which requires phenylalanine or tyrosine Competitive-type inhibition of growth was observed with excess phenylalanine or tyrosine. A major accumulation product recovered was prephenic acid. The new mutant apparently lacks an enzyme involved in the conversion of prephenic acid to phenylalanine and tyrosine.