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Genetic testing for oncology
for the selection of targeted therapy, immunotherapy and in cases of suspected inherited cancer syndromes
Molecular techniques difference in oncology field
Molecular techniques applied in oncology primarily serve to clarify the diagnosis, predict the course of the disease and select the most effective therapy (including targeted drugs).
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NGS – Next-Generation Sequencing
The most innovative and promising techniques comprise next-generation sequencing (NGS), that gives the possibility to sequence both the whole genome or exome, and a set of specific genes. Sequencing of numerous DNA fragments takes place simultaneously that gives high information content at a relatively high speed. Using the NGS method, it is possible to assess at the same time the presence of various genetic disorders in DNA and RNA molecules, such as hotspot mutations (mutations of an increased frequency in the population), single nucleotide variants (SNV), copy number variations (CNV), large insertions , deletions and gene fusions.
A perfect example of using NGS in laboratory practice for oncodiagnostics is Oncomine Focus Assay.
Oncomine Focus Assay is a targeted next-generation sequencing (NGS), a multi-biomarker analysis based on the Ion Torrent platform that determines variants throughout 52 cancer-associated genes.
990 different mutations are identified with Oncomine Focus Assay.
Pros of the method
- A multipurpose analysis for detection of wide range of mutations
- Allows diagnostics without a clear preliminary hypothesis
- Simultaneous analysis of multiple candidate genes
- 10 ng of DNA or RNA is sufficient for the assay
- One of the most sensitive methods
Cons of the method
- Very expensive
- Used for diagnostics of solid tumors only
990 different mutations are found using Oncomine Focus Assay
FISH — fluorescent in situ hybridization
FISH (fluorescent in situ hybridization) — makes it possible to find the exact position of the nucleotide sequences on the DNA or RNA, visualizing them with complementary fluorescent DNA probes. FISH identifies chromosomal rearrangements (deletions, duplications, translocations, inversions). In oncology, it is most relevant when the diagnosis clarification and genome-mutation dependent therapy is needed.
Pros of the method
- Results in a short time
Cons of the method
- Reasonable when suspected of having a mutation on a specific genome location (cannot serve as a screening test)
- Clarification of the biomarker status by other methods is required
- Incapable to identify intrachromosomal disorders, i.e. most types of mutations
- Possibility of false positive results (1–5%)
- High price
Identifies chromosomal mutations: deletions, duplications, translocations, inversions.
arrayCGH — comparative genomic hybridization
arrayCGH (comparative genomic hybridization) is a method that allows detecting unbalanced chromosomal rearrangements (deletions and duplications) across the whole genome. The studied and control DNA are labeled with different fluorescent dyes, mixed and hybridized on microarrays with DNA probes, quantitatively comparing the fluorescence intensity during the protocol.
Pros of the method
- Analysis of pathogenic variants in the whole genome simultaneously
Cons of the method
- Points to the presence of a variant and necessitates the use of additional confirming methods
- Unable to identify balanced chromosomal rearrangements and point nucleotide variants (replacements, deletions, insertions)
- Incoming DNA concentration requirement
- High price
Polymerase Chain Reaction — PCR
PCR (polymerase chain reaction) is a simple but efficient molecular method that enable to multiply copies of certain DNA or RNA fragments for the target fragment qualitative or quantitative evaluation. In oncology, it applies for single nucleotide genetic variants (allele-specific PCR) or microRNA identification. Meanwhile, PCR is frequently a part of more complex diagnostic methods, in particular, sequencing and widely used to determine the presence of potentially carcinogenic pathogens (HPV types 16 and 18, EBV infections, HBV and HCV, retroviruses, etc.).
Pros of the method
- Low-cost
- Available
- Enable to determine the presence of potentially carcinogenic DNA / RNA viral and bacterial agents
- Enable using of any biomaterial containing DNA or RNA
Cons of the method
- False positive results are possible
- In diagnostic laboratories, the testing is usually limited to searching for the most common mutations in a target population.
- Must be combined with other methods for maximum efficiency
It identifies the presence of potentially carcinogenic pathogens HPV types 16 and 18, EBV infection, HBV and HCV, retroviruses, etc.
A brief comparison of some characteristics of the described methods
NGS
FISH
aCGH
PCR
Diagnosis of inherited tumor syndromes
Yes
No
Yes
Yes
Types of tumors
Solid
Solid and hematological
Solid and hematological
Solid and hematological
Biomaterial used
Tissues
Blood, tissues, cellular sediment of urine
Tissues, blood
Any
Limitations
Not suitable for hematological types of tumors testing
The number of mutations studied in a single approach is limited
It is able to identify only large structural variations of the genome
High requirements for the quality of DNA samples provided
Tumor cells content must be above 50%
One analysis examines a limited number of specific mutations in a particular gene