SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis.

DNA Melting Analysis (DMA) with a TaqMan probe covering the mutation "hot spot" is a simple, sensitive, and "closed tube" method of mutation detection. However, DMA requires asymmetric PCR to produce single-stranded amplicons capable of interacting with TaqMan probes. This makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent PCRs. The SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with TaqMan probes. Hybrids of stem-loops and complementary TaqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. In addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. For mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay.

EPSTEIN-BARR VIRUS AND NASOPHARYNGEAL CARCINOMA: VIRAL MARKERS FOR DIAGNOSTICS AND ASSESSMENT OF CLINICAL STATUS OF PATIENTS.

The etiological role of the Epstein-Barr virus (EBV) in the development of an undifferentiated histological variant of nasopharyngeal carcinoma (uNPC) found for the first time in regions with a high incidence of this pathology, the Southern provinces of China and the countries of Southeast Asia, and later in the rest of the world, has served as a basis for the widespread use of EBV serological markers for the diagnosis of this form of tumor. In recent years, the use of a test based on the quantitative determination of the EBV DNA concentration in the blood plasma of uNPC patients for early detection and monitoring of the disease has become widespread in endemic regions. In non-endemic regions, such studies virtually have not been carried out, and moreover, the comparative evaluation of the significance of two viral markers, serological and EBV DNA load in the bloodstream of uNPC patients, for diagnostics and evaluation of the therapeutic effect was not investigated. The aim of this study was to compare the clinical value of two serological markers and plasma EBV DNA load in uNPC patients from non-endemic region (Russia). The obtained results indicate that IgA antibodies to the viral capsid antigen (IgA/VCA) and plasma EBV DNA concentration can be successfully used for the diagnosis of uNPC, while IgG/VCA antibodies have no practical significance as an uNPC marker. In addition, it was found that plasma EBV DNA load is more sensitive marker of uNPC than IgA/VCA titers because DNA copy numbers reflect more accurately the effect of the therapy and the clinical state of patients at the stages of remission or relapse. It was shown for the first time that in the non-endemic region the simultaneous evaluation of IgA/VCA antibody levels and the plasma EBV DNA loads are the most effective markers for the diagnostics of uNPC. However, we believe, that it is more practical to use IgA/VCA antibody levels for uNPC screening, and plasma EBV DNA copies - for monitoring of the disease.

Epstein-Barr virus biomarkers for nasopharyngeal carcinoma in non-endemic regions.

The Epstein-Barr virus (EBV) plays a key role in the development of undifferentiated nasopharyngeal carcinoma (uNPC). In uNPC endemic regions EBV-specific antibodies and plasma EBV DNA load are used as markers for the early detection of uNPC and monitoring of the disease. In non-endemic regions, such studies were practically not conducted. The aim of this study was to compare the clinical significance of EBV serological markers and plasma EBV DNA levels for uNPC patients in a non-endemic region, Russia. The results obtained indicate that both viral capsid antigen/immunoglobulin A (VCA/IgA) antibodies and plasma EBV DNA copies can effectively be used for nasopharyngeal carcinoma (NPC) diagnosis. Besides, plasma EBV DNA load was found to be a more sensitive marker of uNPC than VCA/IgA antibody titres, as it reflected the effect of the therapy in stages of remission and relapse of the disease more precisely. Our study, for the first time, demonstrates that the simultaneous use of plasma EBV DNA loads and VCA/IgA antibody levels are indispensable markers for uNPC in non-endemic regions: a serological marker can be more effectively used for NPC screening, but EBV DNA copies are better for monitoring the disease. However, both markers turned out to be practically unsuitable for assessing the clinical status of patients. Serological markers did not correlate with any signs of the tumour process estimated by tumour, node and metastasis (TNM) classification and the plasma EBV DNA loads correlated only with the size of the pathologically altered lymph nodes (N). Additional study is required to confirm these findings.

[Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

Cancer: Bad Luck or Punishment?

Contrasting opinions on the role of extrinsic and intrinsic factors in cancer etiology (Tomasetti, C., and Vogelstein, B. (2015) Science, 347, 78-81; Wu, S., et al. (2016) Nature, 529, 43-47) variously define priorities in the war on cancer. The correlation between the lifetime risk of several types of cancer and the total number of divisions of normal self-renewing cells revealed by the authors has given them grounds to put forward the "bad luck" hypothesis. It assumes that ~70% of cancer variability is attributed to random errors arising during DNA replication in normal, noncancerous stem cells, i.e. to internal factors, which is impossible either to expect or to prevent. This assumption caused many critical responses that emphasize, on the contrary, the defining role of extrinsic factors in cancer etiology. The analysis of epidemiological and genetic data presented in this work testifies in favor of the "bad luck" hypothesis.

Response to Comments by V. N. Manskikh: "Do External or Internal Factors Lead to Tumor Development? It Is Still Unknown".

The opinion is presented according to which the "bad luck" hypothesis (Tomasetti, C., and Vogelstein, B. (2015) Science, 347, 78-81), which has recently received experimental confirmation, has the right to exist, and its criticisms are largely unfounded.

Cancer research: a hurdle race.

Cancer research has shifted in recent years from studying intracellular processes (identification of damaged genes and signaling pathways) to extracellular (hierarchy of tumor cells, cell transitions, clone competition) and tissue (interactions of a tumor with its environment) research. But then the next step seems to be logical: studying biochemistry of tumor-bearing organisms (namely, cancer-induced changes in cellular and tissue metabolism leading to the organism's death). These data can help to develop new methods of cancer treatment. This article discusses some of the challenges of contemporary oncology and possible ways to overcome them.

[The LMP1 oncogene sequence variations in patients with oral tumours associated or not associated with the Epstein Barr].

The role of the Epstein-Barr virus (EBV), a ubiquitous lymphotropic human herpesvirus type 4, in the etiology of nasopharyngeal carcinoma (NPC) is not fully understood. The mechanism of NPC carcinogenesis, associated with the virus, is also not clear. The objective of present investigation was to carry out comparative analysis of the structure of an LMP1 oncogene of EBV in viral isolates obtained from patients with two types of tumors of the oral cavity: (a) associated (i.e., NPC) and (b) not associated (other tumors of the same anatomical region, OTOC) with EBV. Comparative analysis of C-terminal regions of LMP1 variants that was based on a sequence analysis of LMP1 from tumor, blood and throat washing samples of NPC and OTOC patients showed that all structural characteristics of LMP1 in both groups of patients were genetically similar, and differences found between compared parameters were statistically insignificant. Thus, for the first time it has been revealed that in NPC and OTOC patients in Russia genetically related EBV strains with structurally similar LMP1 variants are persisting that are likely to reflect a polymorphism of the virus circulating in population. The findings allow us to suggest that in non-NPC-endemic regions of the world, which include Russia, the risk of NPC development does not depend on the EBVstrain and its variant of LMP1 so much, but mostly from the genetic predisposition of infected persons to the disease and the exposure to other, as yet unknown agents.

Isotachophoresis of nucleic acids in agarose gel rods.

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.

Carcinogenesis: evolution of concepts.

Cancer is considered as an unintended consequence of internal imperfection of multicellular organisms: Darwinian evolution "does not foresee the future and does not plan for it", it is forced to handle only anything that it has at a given moment "at hand", which makes inevitable compromises and restrictions. In this case, there are a number of founding dogmas including mutagenesis as the main driving force of carcinogenesis; the environment as the main source of mutagenic effects; tumor monoclonality; cancer cell multistage transformation as Darwinian process of successive mutation-selection cycles. Recent discoveries complicate, supplement, and sometimes transform into an opposite fixed concepts. As a result, a new "image" of carcinogenesis is formed as a biological phenomenon whose conservation is indicative of its evolutionary utility.

Cancer as a programmed death of an organism.

The hypothesis introduces the idea that there is a critical level of mutagenesis that triggers a program of organism death by means of proliferation of killer cells. Similarly to apoptosis, which is an altruistic suicidal act of a faulty cell threatening the stability of a multicellular organism, a malignant tumor is an altruistic suicide of an individual carrier of harmful alleles threatening genetic stability of the population.

Circulating nucleic acids and apoptosis.

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.

Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.

DNA methylation and carcinogenesis.

In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).

Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains.

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes.

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.

Rearrangements of DNA-protein interactions in animal cells coupled with cellular growth-quiescence transitions.

Overall DNA-protein interactions in animal cells undergo drastic changes coupled with cellular transitions from quiescence to growth and reversely as revealed by nucleoprotein-Celite chromatography. DNA of chromatin was found to exist in one of the two sharply distinct alternative forms, namely, either tightly or weakly bound to protein moiety. These forms are specific for cycling and quiescent cells, respectively. The tight DNA-protein interactions characterize all cycling cells independent of the cell cycle phase. Transition of DNA of cycling cells from one form to another was observed as a result of treatment of isolated nuclei with DNase I.