A wide variety of tests are performed in the laboratory for accurate pathologic diagnosis and assessment of tumor biomarkers. These tests range from immunohistochemistry to determine expression of proteins within cells to a whole host of molecular tests evaluating tumor DNA and RNA characteristics. Methodology and examples of utility for the most commonly used tests are summarized in the following section.
Immnohistochemistry (IHC) is performed routinely in pathology laboratories to determine the expression of proteins within a tissue specimen.
- Tissue is fixed and prepared on a microscope slide.
- The tissue is first exposed to the primary antibody. This is the antigen-specific antibody.
- The tissue is next exposed to the secondary antibody. The secondary antibody is typically developed to bind to the first one by using a general species-specific antibody created against the immunoglobulin G of the species in which the primary antibody was made. For example, if an antibody was made in a mouse or in mouse cells in culture, then the secondary antibody would be anti-mouse. This antibody binds to the primary antibody and serves two purposes:
- Signal amplification: Often, a number of secondary antibodies bind to a single primary antibody; thus, for every single antigen-primary antibody binding event, multiple secondary antibody-primary antibody binding events occur, thus amplifying the initial binding event.
- Signal detection: Commonly, a reporter is conjugated to the secondary antibody. This reporter molecule is used to monitor the IHC reaction.
For example, the peroxidase enzyme may be conjugated to the secondary antibody. Upon addition of the substrate diaminobenzidine (DAB), the peroxidase enzyme catalyzes its oxidation, resulting in the production of a brownish colored product that can be visualized under an ordinary microscope.
- As another example, a fluorophore such as fluorescein may be conjugated to the secondary antibody, which may be visualized using a fluorescent microscope.
- By using a secondary antibody — termed indirect detection — the cost is reduced by avoiding having to conjugate every antibody to an enzyme for detection (known as direct detection); only general secondary antibodies need this done.
- Immunohistochemistry can give semi-quantitative results as well as cellular location (eg, cytoplasm, nucleus, membrane) of the protein of interest. Typically, IHC is quantified by percent of cells that are positive for the stain and intensity on a scale of +1 to +3. If a protein is found by this method, then it is termed IHC positive, and the protein is considered over-expressed.
- Tissue processing and specimen fixation (pre-analytical variables) can significantly affect the results. Standardization of pre-analytical variables helps ensure accurate and reproducible results.
- For example, over-fixation can destroy the epitope on some antigens, rendering it unrecognizable by the antibody.
- Decalcification agents (eg, hydrochloric acid) used on bone or other calcified tissues may result in decreased antigenicity of tissue.
- Antibodies can be characterized by their specificity, sensitivity and affinity.
- Visual interpretation can be subjective and differs between pathologists. Computerized image analysis of IHC staining (eg, scoring of HER-2 in breast cancer) has removed some of the subjectivity from IHC.
Example applications in evaluation of tumors
- Categorization of malignant tumor type (eg, carcinoma, lymphoma, germ cell, sarcoma, melanoma): It may not be possible to classify certain tumors based solely on their histologic appearance.
- Determining site of origin for metastatic tumors.
- Subclassification of tumor in various organ systems (eg, specific subtype of lymphoma).
Example applications in oncology biomarkers
- Estrogen receptor/progesterone receptor staining (breast cancer): used to determine if a patient with breast cancer is hormone receptor positive; these patients will be candidates for treatment with tamoxifen or aromatase inhibitors.
- HER-2 (breast cancer): used to determine if a patient with breast cancer will be a candidate for HER-2–directed therapy, most notably trastuzumab (Herceptin, Genentech), lapatinib (Tykerb, Novartis), pertuzumab (Perjeta, Genentech) and trastuzumab emtansine (Kadcyla, Genentech).
- PD-L1 (non–small cell lung cancer): used to determine if a patient with metastatic NSCLC may be a candidate for pembrolizumab (Keytruda, Merck).
In situ hybridization
In situ hybridization (ISH) is a laboratory test that allows for the precise localization of a specific region of nucleic acid on a chromosome or in tissue.
- Uses a sequence-specific DNA, RNA or oligonucleotide probe to bind to a complementary section of the DNA or RNA.
- Visualization of a reporter molecule on the probe allows for the detection and localization of a particular nucleic acid target within a heterogenous cell population, such as a tissue sample.
- Used to evaluate genetic abnormalities, including amplifications (as opposed to over-expression by changes in translational or post-translational events), deletions, translocations and aneuploidy.
- Most ISH procedures use a fluorescently-labeled probe. This is referred to as fluorescent in situ hybridization (FISH). FISH studies are evaluated under a fluorescent microscope. Alternatively, when a chromogenic probe is used, it is referred to as chromogenic in situ hybridization (CISH). CISH studies are evaluated using a bright-field microscope.
- Within the sample, the target DNA is first denatured with either heat and/or chemicals. This step is necessary to create a single-stranded template that will hybridize to the probe.
- The labeled probe is then added to the sample. The probe hybridizes, or binds, to the target sequence in a sequence-dependent fashion.
- Probe-bound DNA is visualized using a fluorescent microscope (or a bright-field microscope in the case of CISH).
- FISH is amenable to multiplexing when multiple targets are visualized in the same sample by using different fluorophores to label the probe.
Example applications in oncology biomarkers
- Anaplastic lymphoma kinase gene fusions: FISH tests can be used to determine if a fusion of the ALK gene (most commonly to the EML-4 gene) is present in patients with metastatic non–small cell lung cancer . If so, then these patients are candidates for treatment with ALK-targeted therapies, including crizotinib (Xalkori, Pfizer), alectinib (Alecensa, Genentech), and ceritinib (Zykadia, Novartis). The most common FISH test for ALK rearrangement includes two differently colored (red and green) fluorescent probes that flank the highly conserved translocation breakpoint within ALK. In non-rearranged cells, the red and green probes are in close proximity, resulting in a yellow (fused) signal. In patients with an ALK rearrangement, these probes are separated, resulting in a space between the red and green signals, which then fluoresce independently in color.
- Epidermal growth factor receptor amplification: EGFR gene amplification can result in overexpression of the EGFR cell surface receptor, a target for inhibition by either small molecule inhibitors (including erlotinib [Tarceva; Genentech, Astellas Oncology] and gefitinib [Iressa, AstraZeneca]) or by anti-EGFR antibodies (such as cetuximab [Erbitux, Eli Lilly], panitumumab [Vectibix, Amgen] and necitumumab [Portrazza, Eli Lilly]). EGFR amplification has been associated with a favorable clinical benefit to targeted EGFR inhibition in both NSCLC and colorectal cancer.
- HER-2 amplification: HER-2 status of breast and gastric/gastroesophageal junction carcinomas may be assessed by in situ hybridization. These studies typically use two separately colored probes, one for the HER-2 gene and one as the control probe, to assess for HER-2 gene amplification.
Polymerase chain reaction
Polymerase chain reaction (PCR) is a laboratory method, developed by Kary B. Mullis, PhD, in which a target DNA template sequence is amplified several orders of magnitude. This technique is used in many fields in addition to oncology, including cloning, forensics and infectious disease. For more information on PCR as it relates to immuno-oncology, see the section "Types of Molecular Testing -- Clinical."
- Original versions of PCR, known as end-point PCR, required the user to separate the amplified DNA products by size on an agarose gel electrophoresis.
- Current clinical versions of PCR are real-time PCR, which include the use of a fluorescently labeled sequence-specific probe to monitor the production of a specific amplified product in real time during the reaction. This can be used quantitatively (termed quantitative PCR or qualitatively. Digital PCR is another quantitative approach to PCR using a very diluted DNA template, and many PCRs run in parallel with the proportion of negative results used to calculate the DNA concentration.
- RNA may be used as the template in the reaction, when it is first reverse transcribed to a complementary DNA sequence. This form of PCR is termed reverse transcription PCR.
- Typically, a purified DNA sample is required. This means that the DNA must first be extracted from the tumor specimen.
- The PCR process is a thermocyclic process, meaning that it proceeds through changes in temperature in a repeated fashion. Many real-time PCR assays use a three-step cycling method.
- In the first step, denaturation, the DNA is heated to a high temperature (at or near 98°C). This step separates the DNA strands, turning the double-stranded DNA sample into a single-stranded DNA template.
- In the second step, annealing, the temperature is lowered to below the melting temperature of the sequence. This allows two primers (referred to as the forward and reverse primers), to bind to the complementary sequence within the target DNA. Usually, these primers are within 100 bp and 500 bp apart.
- In the third step, elongation, a DNA polymerase (often, Taq DNA polymerase) is used to synthesize new DNA sequence. The polymerase sits down on the DNA at the 3’ end of each primer and travels along the template strand, adding new DNA nucleotides in a sequence-specific manner.
- The product of each cycle is the generation of a new double-stranded DNA amplicon that is identical to the target sequence within the template strand.
- This process is repeated many (35 to 55) times.
- Often, the second and third step are combined to occur at a single temperature, which may speed up the overall PCR reaction.
- In real-time PCR, a sequence-specific probe is included in the reaction. The probe is labeled with a fluorophore on one end and a quencher on the other. When the probe is not bound to the target sequence, the fluorescence of the fluorophore is effectively quenched by the quencher. When the probe is bound to the target sequence, the exonuclease activity of the polymerase cleaves the fluorophore off the probe as it travels along the DNA template. This increases the distance of the fluorophore from the quencher, thereby causing a measurable increase in the fluorescent signal.
- An important consideration in any PCR reaction is the DNA polymerase used. High-fidelity DNA polymerases have lower error rates but are also more expensive. Because of the amplification, errors can also be greatly amplified using PCR.
- Contamination is also an issue because of the ability to amplify a single DNA molecule and detect it. Consequently, many labs have clean areas to separate DNA isolation and PCR amplification or the use of special laminar flow hoods to protect the sample from contamination.
- Finally, the design of the oligonucleotide primers is a critical step to ensure specificity and amplification of the correct region of DNA.
Example applications in oncology biomarkers
- Epidermal growth factor receptor mutation testing: Real-time PCR is used to monitor for the presence or absence of certain mutations within the EGFR gene in patients with NSCLC. These mutations are known to sensitize the tumor to treatment with EGFR tyrosine kinase inhibitors such as erlotinib (Tarceva; Genentech, Astellas Oncology) and gefitinib (Iressa, AstraZeneca).
Sequencing is a method to determine the order and type of nucleotide within a DNA molecule. It is an alternative way to determine genetic alterations.
- Advantageous over quantitative polymerase chain reaction (PCR) because it detects all mutations present, not just known mutations.
- Provides a high-resolution, base-by-base view of the entire genome, exomes, targeted genes or hotspots, depending on the sample preparation.
- Captures both large and small abnormalities that might otherwise be missed including point mutations, small insertions or deletions of nucleotides (called indels), frameshifts, fusions between genic regions and amplification or loss of genes or regions of genes.
- Creating personalized plans to treat disease may be possible based not only on the mutant genes causing a disease, but also on other genes in the patient’s genome.
- Genotyping cancer cells allows physicians to select the best treatment and potentially expose the patient to less toxic treatment because the therapy is tailored (also known as personalized, individualized or precision medicine).
- Previously unknown genes may be identified as contributing to a disease state; whereas traditional genetic testing looks only at the common “troublemaker” genes.
- Disadvantages are cost and time; however, the price continues to decrease, and the time to results continues to shorten.
- The role of most genes in the human genome is still unknown or incompletely understood; therefore, a significant amount of the information found in a human genome sequence is currently unusable.
- Most physicians are not trained in how to interpret genomic data. The rate of new findings is rapid, and it is challenging stay current.
- An individual’s genome may contain information that they do not want to know. For example, a patient may have genome sequencing performed to determine the most effective treatment plan for high cholesterol. In the process, researchers discover an unrelated allele that assures a terminal disease with no effective treatment. Thus, it is important to inform the patient of the potential results before testing.
- The volume of information contained in a genome sequence is vast. Policies and security measures to maintain the privacy and safety of this information are still new.
- Traditional chain-termination sequencing methodology that was developed in 1977, also known as dideoxy sequencing.
- Method has been updated and is conducted by automated sequencers using a modification of the original method, in which a different color dye is used for each nucleotide and capillary electrophoresis is used in place of the gel electrophoresis of the original method.
- Although Sanger sequencing was widely used for more than 25 years, it is rarely used now due to the increased sensitivity and reduced costs of next-generation methods.
- Target sequence-based method that allows rapid quantification of sequence variation using detection of pyrophosphate release as nucleotides are incorporated in a sequencing-by-synthesis mode.
- Target section of DNA is first synthesized by hybridizing the template to a primer and incubating with necessary reagents releasing a pyrophosphate. This is converted to a luciferase-catalyzed reaction, which is detected by a camera creating a pyrogram that is interpreted to determine the sequence.
- A limitation is the shorter length of the individual reads obtained as compared with Sanger sequencing, leading to more complicated assembly.
- Next-generation sequencing is performed by a process similar to capillary electrophoresis, but instead of sequencing a single DNA sequence, next-generation sequencing extends the process across millions of fragments of DNA in a parallel fashion, greatly speeding up the sequencing and decreasing the cost. This approach requires complicated reassembly, alignment and bioinformatics analysis to obtain the final sequence. Multiple technology platforms are considered next-generation sequencing.
- Next-generation sequencing requires three steps: sample preparation, sequencing and data analysis.
- Sample preparation requires two steps:
- Library preparation: Sample preparation requires creation of a library. Many nuances and approaches to library preparation are available, depending on the next-generation sequencing platform used, but the goal is the same requiring random fragmentation and tagging of DNA with adaptors. Tags with specific known sequences, known as barcodes, may be used to enable sample pooling, decreasing the cost, increasing efficiency and aiding in assembly after sequencing. Targets may be enriched using multiplexed PCR or hybridization to isolate the regions of interest before sequencing (eg, hotspots [specific base-pairs], targeted exons of genes, whole exome).
- Cluster amplification is used to create clonal clusters of a library. After denaturing and hybridization to immobilize within the next-generation sequencing flow cell, the DNA is amplified. Repeating this many times creates the cluster.
- Sequencing: There are several major next-generation sequencing platforms for sequencing.
- Sequencing by synthesis: Advances in sequencing by synthesis transformed pyrosequencing to a next-generation method. DNA samples are cleaved into short fragments (about 100 bp to 150 bp), and then adapters are ligated to these fragments for PCR to be carried out, creating many copies of the same read in a single spot. Taking an image after flooding with fluorescently-labeled nucleotides, DNA polymerase and a terminator to add a single base at a time, enables the sequence to be obtained. An advance to this method is called paired-end sequencing, which incorporates sequencing from both ends of the DNA fragment and aligning both the forward and reverse reads, improving accuracy. Illumina, a company that manufactures next-generation sequencing equipment, uses this type of technology.
- Ion semiconductor sequencing: Another method of sequencing by synthesis uses detection of hydrogen ions released during polymerization on a semiconductor chip to obtain the sequence directly. A single nucleotide is released at a time, and a signal is detected if it is incorporated. The signal is proportional to the number of same nucleotides in a row (eg, three T nucleotides corresponds with three times a single T). This method also uses DNA fragments, but the size is a little larger (about 200 bp). Thermo Fisher Scientific’s Ion Torrent platform and Ion Proton system use this approach.
- Single-molecule real-time sequencing: Pacific Biosciences uses a different technology, parallelized single-molecule, real-time technology, to perform next-generation sequencing. In this approach, the DNA polymerase is immobilized with a single DNA molecule template. Fluorescent-labels are cleaved off of the nucleotides as they are incorporated, providing a signal for detection and determination of the sequence. This approach has much longer read lengths making alignment and assembly much easier.
- Data analysis: Data analysis for next-generation sequencing typically has multiple steps in the process often referred to as a "pipeline," as it is standardized for each analysis type. Here are the major steps typically found in most analysis pipelines:
- After the bases are called in each of the reads, the reads are aligned to a reference genome to assemble the gene sequence. Duplications are removed, leaving unique reads only.
- Single-nucleotide polymorphisms (SNPs) must be identified before calling variants to remove population heterogeneity.
- Variants, including small insertions/deletions (indels), are called subsequently by comparison to the reference genome after identifying SNPs. The accuracy of variant calling depends on the depth of coverage at each base. Depth is how many times a base is sequenced from unique reads (after removal of duplications).
- Annotation of the variants called typically relies on a number of databases to note whether the variant is a known pathogenic or benign variation or one whose function is still unknown (variant of unknown significance). A number of companies have created extensive custom data systems to annotate the alterations identified. Publically available databases are also often used (eg, the Short Genetic Variations database [dbSNP], HapMap (decommissioned and archived), 1000 Genomes Project, Catalogue of Somatic Mutations in Cancer [COSMIC], ClinVar, Online Mendelian Inheritance in Man [OMIM]).
Next-generation sequencing technology can also be used with RNA to look at the transcriptome or expression of genes. Often this is referred to as RNA-Seq, or RNA sequencing. Other modifications enable analysis of the methylome.
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