Molecular profiling has become an essential tool in cancer research and the clinical management of cancer patients. Targeted and personalized therapies are now commonplace in clinical practice, tailored to the specific genetic or protein biomarkers that underlie and drive a patient’s tumor growth, metastasis, treatment resistance, and recurrence.1
In the research world, a detailed understanding of the biomarkers detected at the various stages of cancer development can be used to develop new treatments, diagnostic tests, and a better understanding of the biology of cancer.
Below, we take a closer look at molecular profiling in cancer research and some of the key steps and considerations for molecular profiling workflows.
What is Tumor Molecular Profiling?
Tumor molecular profiling is a method that detects the presence of specific biomarkers – genes, proteins, or other molecules – in tumor tissue or blood sample.1 There are a variety of tumor molecular profiling methods that are used in cancer research and oncology, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), PCR, and next-generation sequencing (NGS) of DNA and/or RNA, which can uncover certain genetic mutations or molecular changes.1
As PCR- and NGS-based approaches to tumor profiling have become cheaper and the number of genetic alterations associated with specific clinical outcomes has increased, they have usurped conventional techniques such as IHC and FISH. These modern techniques have gained steam over the past decades as they enable a multiplexed analysis of multiple loci, whereas traditional methods focus on a single target. With faster workflows, more sophisticated bioinformatics analysis, robust publicly available databases, and comprehensive tumor banks, PCR and NGS have become the favored methods for cancer research and precision medicine.
The 4 Steps in Tumor Molecular Profiling Workflows
With PCR and NGS in the driver’s seat as the preferred techniques for molecular profiling of tumors, there’s a significant need for nucleic acid extraction kits, reagents, and streamlined workflows for generating and analyzing results. Yet, choosing which platforms and reagents are appropriate for a specific downstream application remains challenging. To better understand what’s needed for these workflows, let’s look at each step in the process of molecular profiling of tumors.
Step #1: Sample Collection
The first step in PCR- or NGS-based molecular profiling is sample collection. In cancer research, DNA or RNA extraction from tissue biopsies or solid tumors is standard. However, there can be several downsides to using these types of samples: Due to the invasive nature of sample collection, limited, single timepoint samples are available, providing only a single snapshot to analyze the tumor and assess tumor heterogeneity. In addition, some tissue samples may be formalin-fixed, paraffin-embedded (FFPE), which causes damage to the nucleic acids that you may be trying to analyze.
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 enable researchers to take multiple samples over time, allowing longitudinal tracking of cancer evolution. Different biomolecules can be detected from these samples, including DNA, RNA, protein, or metabolites; however, cell-free DNA is commonly used for downstream NGS applications.2
Step #2: Sample Preparation
For PCR- or NGS-based molecular profiling, sample preparation typically involves the extraction of nucleic acids from the sample types discussed above. As cancer research has grown, the number of protocols and commercially-available kits for DNA, RNA, or both has expanded. There are specialized DNA and RNA extraction kits for FFPE samples, total blood, and those that can specifically isolate microRNAs. Ultimately, what DNA/RNA extraction method or kit you use will be informed by your sample type, downstream application, and the basic biological question you’re trying to answer.
Another consideration for your DNA or RNA extraction is whether this step will be performed manually or using automated instrumentation. There are several commercially-available platforms for automated sample preparation, helping bring consistency, safety, and many other benefits to the overall sample extraction process.
Step #3: Molecular Analysis
Isolated DNA or RNA can be used for several molecular profiling techniques. Quantitative real-time PCR (RT-PCR) has long been a favorite amongst cancer researchers as a rapid, highly sensitive, and specific technique for multiplexed mutation detection and gene expression analysis. Though RT-PCR still has its place, many researchers also employ digital PCR (dPCR) platforms.3
dPCR doesn’t have the multiplex capabilities of RT-PCR but does provide sensitive and specific absolute quantification of a genomic mutation or transcript without the need for standard curves or normalization, commonly used in RT-PCR protocols. And with the increasing application in cancer research – for both liquid and tissue biopsies – there are many platforms and reagents available for dPCR.
NGS is another molecular technique that provides several advantages over PCR-based methods, namely the ability to detect a range of different 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 massive amount of data, a significant benefit for exploratory studies focused on furthering our understanding of cancer biology.
NGS can also be used to analyze targeted gene panels, which can include tens to hundreds of genes. These are more commonly used in clinical settings as they offer greater coverage in specific regions, quicker results, and more relevant data than WES or WGS methods.
One final 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 large 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 in most molecular profiling workflows is to analyze data outputs from your PCR- or NGS-based assays. For RT-PCR or dPCR, most instruments will provide tailored software that can help transform raw data into a publication-ready figure.
There are more options for NGS data: If your team includes a skilled bioinformatician who can build and run a data analysis pipeline, you can quickly process and visualize your data. There are many open-access tools and databases to choose from, and what you employ will largely depend on your sequencing technique or platform. Much like the PCR-based methods above, commercial platforms will have their own software suites to help make visualizing results more streamlined and straightforward for those researchers who are not bioinformatics experts.
End-to-End Molecular Profiling Workflows for Cancer Research
Thermo Fisher Scientific offers a complete suite of molecular profiling solutions for cancer researchers analyzing liquid biopsy and solid tumor samples. Our magnetic bead and silica spin column-based kits can accommodate a wide range of sample types, including FFPE tissue samples, plasma, blood, and serum in automation-compatible formats. In addition, our proven RT-PCR, dPCR, and NGS instruments provide sensitive and precise assays that deliver you with the depth of data needed to help drive your research forward.
Learn more about our end-to-end cancer workflows here.
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This article is for Research Use Only. Not for use in diagnostic procedures.
References:
- Malone ER, Oliva M, Sabatini PJB, Stockley TL, Siu LL. Molecular profiling for precision cancer therapies. Genome Med. 2020;12(1):8. doi:10.1186/s13073-019-0703-1.
- A complete next-generation sequencing workflow for circulating cell-free DNA isolation and analysis. Thermo Fisher Scientific website: https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/cfDNA-appnote.pdf. Accessed January 23, 2023. Published October 26, 2015
- Coccaro N, Tota G, Anelli L, Zagaria A, Specchia G, Albano F. Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies. Int J Mol Sci. 2020;21(9):3141. Published 2020 Apr 29. doi:10.3390/ijms21093141
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