Aberrant methylation scanning by quantitative DNA melting analysis with hybridization probes as exemplified by liquid biopsy of SEPT9 and HIST1H4F in colorectal cancer.

OBJECTIVE: The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable. METHODS: The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed. qDMA-HP is characterized by the following features: (i) asymmetric PCR with methylation-independent primers; (ii) fluorescent dual-labeled, self-quenched probes (commonly known as TaqMan probes) covering several interrogated CpGs; (iii) post-PCR melting analysis of amplicon/probe hybrids; (iv) quantitation of unmethylated and methylated DNA alleles by measuring the areas under the corresponding melt peaks. RESULTS: qDMA-HP was tested in liquid biopsy of colorectal cancer by evaluating SEPT9 and HIST1H4F methylations simultaneously in the single-tube reaction. Differences in the methylation levels in healthy donors versus cancer patients were statistically significant (p < 0.0001), AUCROC values were 0.795-0.921 for various marker combinations. CONCLUSIONS: This proof-of-concept study shows that qDMA-HP is a simple, normalization-independent, quantitative, multiplex and "closed tube" method easily adapted to clinical settings. It is demonstrated, for the first time, that HIST1H4F is a perspective marker for liquid biopsy of colorectal cancer.

Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer.

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.

Genetic Mosaicism and Cancer: Cause and Effect.

Increasing theoretical and experimental evidence suggests that the genomes of both normal and cancer cells are subject to continuous changes as a result of copying errors during replication, defects in chromosome segregation during mitosis, and direct chemical attacks by reactive oxygen species. The process of cellular genetic diversification begins during embryonic development and continues throughout life, leading to the phenomenon of somatic mosaicism. New information about the genetic diversity of cells composing the body makes us reconsider the existing concepts of cancer etiology and pathogenesis. Here, I suggest that a progressively deteriorating microenvironment ("soil") generates the cancerous "seed" and favors its development. Cancer Res; 78(6); 1375-8. ©2018 AACR.

TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.

Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved.

Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations.

The data in this article are related to the research article entitled "Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations" Botezatu et al. [1]. Somatic mutations in the PIK3CA gene ("hot spots" in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.

Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations.

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5-3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity-all of the qualities required for clinical genotyping.

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS.

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the "closed tube" format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the "open tube" format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5-SYBR Green I and Bio-Rad CFX96-EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a "lower level" into the range of those suitable for mutation scanning.

Tube gel isotachophoresis: a method for quantitative isolation of nucleic acids from diluted solutions.

The technique of isotachophoresis is intended for separation of molecules having different electrophoretic mobilities in a nonhomogeneous electric field. Since the mobility of nucleic acids in water solutions is uniform and does not depend on their size (because of a uniform distribution of negatively charged phosphate groups along the molecule), isotachophoresis will concentrate rather than separate them in the mobile borderline zone between the rapid (Cl(-)) and the slow (ß-alanine(-)) anions. This idea served as the basis for elaboration of a novel method for isolation of nucleic acids from diluted solutions. Advantages of the method include quantitative yield (regardless of molecule size), high degree of concentration, and the ability to visually monitor the process. The method may find applications in nucleic acid isolation from highly degraded forensic and clinical samples, from bodily fluids in particular, and thereby promote development of this important direction of diagnostics.

Cancer: evolutionary, genetic and epigenetic aspects.

There exist two paradigms about the nature of cancer. According to the generally accepted one, cancer is a by-product of design limitations of a multi-cellular organism (Greaves, Nat Rev Cancer 7:213-221, 2007). The essence of the second resides in the question "Does cancer kill the individual and save the species?" (Sommer, Hum Mutat 3:166-169, 1994). Recent data on genetic and epigenetic mechanisms of cell transformation summarized in this review support the latter point of view, namely that carcinogenesis is an evolutionary conserved phenomenon-a programmed death of an organism. It is assumed that cancer possesses an important function of altruistic nature: as a mediator of negative selection, it serves to preserve integrity of species gene pool and to mediate its evolutionary adjustment. Cancer fulfills its task due apparently to specific killer function, understanding mechanism of which may suggest new therapeutic strategy.

Cancer: shift of the paradigm.

Cancer is usually considered to be a by-product of design limitations of a multicellular organism and its intrinsic fallibility. However, recent data prompt a revision of some established notions about carcinogenesis and form a new paradigm of carcinogenesis as a highly conserved biological phenomenon - a programmed death of an organism. This altruistic program, which is unleashed when mutagenesis surpasses a certain critical threshold, gives a population the important benefit acting as a guardian of the gene pool against the spread of certain mutant genes. A growing body of evidence supports this point of view: (i) epigenetic changes leading to cancer arise early, simultaneously in many cells and look like deterministic regulation; (ii) concept of cancer stem cell suggests a view of carcinogenesis not as vague transformation but as well known differentiation; (iii) tumor/host relations usually perceived as antagonistic are, in reality, synergistic; (iv) death of an individual from cancer is predetermined and results apparently from a specific activity (killer function) of cancer cell and (v) evolutionary conservation indicates that cancer comes with a general advantage that explains its evolutionary success. A holistic approach to carcinogenesis suggests new avenues of research and new therapeutic strategy.

Novel applications of polymerase chain reaction to urinary nucleic acid analysis.

DNA fragments from cells that have died throughout the body not only appear in the bloodstream but also cross the kidney barrier into the urine. The relatively low molecular weight (150-200 bp) of this Transrenal DNA should be considered when deciding on methods of isolation and analysis. In particular, if polymerase chain reaction (PCR) is used for amplification and detection of specific sequences, then the reduction of amplicon size will significantly enhance sensitivity. Detection of DNA mutations is also made more difficult by the presence of a large excess of a wild-type allele. Using K-RAS mutations as an example, two ways around this problem--enriched PCR and stencil-aided mutation analysis-are described, based on selective pre-PCR elimination of wild-type sequences.

Concentration and isolation of DNA from biological fluids by agarose gel isotachophoresis.

Isotachophoresis is an electrophoretic method of separation of charged substances. The method is characterized by a discontinuous buffer system, constant velocity of separated molecules, and the distribution of separated components in the form of narrow concentrated bands located one right after another. As a rule, isotachophoresis is not used for the separation of nucleic acids because the mobility of polynucleotides in this system does not depend on their size. However, this circumstance proved to be very useful for the quantitative isolation of heterogeneous DNA fragments from biological fluids, for gene diagnostics of cancer in particular. The proposed method of agarose gel isotachophoresis of DNA has been used for the isolation of blood DNA and its successful PCR analysis.

On evolutionary origin of cancer.

BACKGROUND: The necessary and sufficient capabilities of cancer cell have been identified. Strikingly, this list does not include one that would seem to be a key property, namely the ability of cancer cells to kill their "host". This is believed to be a self-evident consequence of the other capabilities (e.g., metastasis), although the available evidence suggests a distinct killer function. Taking into account this unlisted property can significantly affect the current paradigm of carcinogenesis. PRESENTATION OF THE HYPOTHESIS: On the assumption that killer function is a key capability of the cancer cell, it is suggested that cancer has evolved as a mechanism of negative selection of mutant alleles of vitally important genes present in population. Similarly to apoptosis, which is an altruistic suicidal act of a damaged cell, cancer is an altruistic suicidal act of an individual who carries dangerous alleles and presents a hazard for genetic stability of the population. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively. TESTING THE HYPOTHESIS: The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function of cancer cell as well as those specific targets in the body that are most strongly affected by the tumor growth. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis suggests new avenues of cancer research as well as principally new therapeutic strategies.