Introduction:
Molecular profiling is an emerging tool for cancer research and clinically managing patients dealing with cancer. Targeted and personalized therapies are commonly used in clinical practice, targeted to the specific genetic or protein biomarkers that underlie and control tumor growth, metastasis, treatment resistance, and recurrence. A few examples of molecular profiling technologies are immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and quantitative polymerase chain reaction (qPCR).
What Is Molecular Profiling?
Molecular profiling is a diagnostic method in which a sample of tissues, blood, or other body fluids is examined to check for genes, proteins, or other molecules indicative of a condition such as cancer. Some of the types of molecular profiling methods include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and quantitative polymerase chain reaction (qPCR). These methods have the potential to detect specific genetic mutations or molecular changes.
As PCR- and NGS-based approaches to tumor profiling have become less expensive and the number of genetic alterations linked with specific clinical outcomes has risen, they have usurped conventional techniques such as IHC and FISH. These modern techniques have gained more popularity during the past decades as they enable a multiplexed analysis of multiple loci, whereas traditional methods aim at a single target. With faster workflows, more sophisticated bioinformatics analysis, robust publicly available databases, and comprehensive tumor banks, PCR and NGS have become favorable methods for cancer research and precision medicine.
What Are the Steps Involved in Molecular Profiling of Tumor?
With PCR and NGS (next-generation sequencing) being the most preferred techniques for molecular profiling of tumors, there is a huge demand for nucleic acid extraction kits, reagents, and streamlined workflows for generating and evaluating the outcomes. The steps involved in the molecular profiling of tumors include the following:
Step 1: Collection Of the Sample
The first step in PCR- or NGS-based molecular profiling is sample collection. DNA or RNA extraction from tissue biopsies or solid tumors is ideal in cancer research. However, there can be several difficulties in using these types of samples due to the invasive nature of sample collection. Limited timepoint samples are available, providing only a single snapshot to analyze the tumor and assess tumor heterogeneity. In addition, some tissue samples might be formalin-fixed, paraffin-embedded (FFPE), which can damage the nucleic acids to be analyzed.
In response to these challenges, liquid biopsies, which can detect circulating tumor-specific biomarkers in blood, plasma, or serum, have emerged as a promising, non-invasive alternative sample type for tumor molecular profiling. They also allow the researchers to take multiple samples over time, allowing longitudinal tracking of cancer evolution. These samples can detect various biomolecules, including DNA (deoxyribonucleic acid), RNA (ribonucleic acid), protein, or metabolites. However, cell-free DNA is usually used for downstream NGS applications.
Step 2: Preparation Of the Sample
For PCR- or NGS-based molecular profiling, sample preparation usually involves the extraction of nucleic acids from the sample types discussed above. As cancer research has expanded, the number of protocols and commercially-available kits for DNA, RNA, or both has increased. There are specialized DNA and RNA extraction kits for FFPE samples, total blood, and those that can particularly isolate microRNAs. Ultimately, what DNA or RNA extraction method or equipment is used will be informed by the sample type, downstream application, and the basic biological question to be answered.
Another consideration for DNA or RNA extraction is whether this process will be performed manually or using automated instrumentation. Numerous commercially available platforms for automated sample preparation assist in bringing consistency, safety, and many other benefits to the overall sample extraction process.
Step 3: Molecular Analysis
Isolated DNA or RNA can be used for numerous molecular profiling techniques. Quantitative real-time-PCR (RT-PCR) has been the most used method for a long time amongst cancer researchers as a rapid, highly sensitive, and specific method for multiplexed mutation detection and gene expression analysis. Though RT-PCR still has its place, many researchers also consider digital PCR (dPCR) platforms.
dPCR does not have the capabilities of RT-PCR but does provide sensitive and specific absolute quantification of a genomic mutation or transcript without the requirement for standard curves or normalization, commonly used in RT-PCR protocols. With the increasing application in cancer research for both liquid and tissue biopsies, various platforms and reagents are available for dPCRPCR.
NGS is another molecular technique with several advantages over PCR-based methods, like detecting various genomic or transcriptomic aberrations, including mutations, copy number variations (CNV), genome translocations, gene fusions, and alternative splice variants. In cancer research, whole exome sequencing (WES) or whole genome sequencing (WGS) provides a huge amount of data, a significant benefit for exploratory studies focused on furthering our understanding of cancer biology.
NGS can also analyze targeted gene panels, including tens to hundreds of genes. These are usually used in clinical settings as they offer larger coverage in specific regions, quicker results, and more relevant information than WES or WGS methods.
One more consideration for NGS approaches is whether or not to use automated instrumentation for library preparation. NGS platforms have exploded in the past decade, and there has been a massive increase in the number of platforms that bring an automated, and thus, more consistent and cost-effective library preparation option to manual workflows.
Step 4: Data Analysis
The final step usually considered in most molecular profiling workflows is to analyze data outputs from the PCR or NGS-based assays. For RT-PCR or dPCR, most instruments will present tailored software that can assist in transforming raw data into a publication-ready figure.
There are more options for NGS data: If the team includes a skilled bioinformatician who can help to build and run a data analysis pipeline, one can easily process and visualize the data. There are various open-access tools and databases to choose from, and what is employed will rely more on the sequencing technique or platform. Much like the PCR-based methods above, commercial media will have their software suites to assist in visualizing the results more streamlined and straightforward for those researchers who are not bioinformatics experts.
Conclusion:
Molecular therapies are used to detect and treat cancer, which has shown great results in cancer treatment. PCR and NGS (next-generation sequencing) were the most preferred techniques for molecular profiling tumors, but new techniques are also being developed. Molecular profiling involves a few steps, such as selecting the sample, preparing the sample, molecular analysis, and data analysis.
