Biochemical testing

This type of testing measures the level of a biomarker, usually a protein, that is secreted by the tumor cells into the blood or other body fluids such as urine or cerebral spinal fluid. The healthcare provider/oncologist orders the test and the appropriate sample is collected from the patient. Testing for the majority of biochemical biomarkers does not require insurance approval. The result of the test is reported as a number and is interpreted as abnormal if outside of the normal range of expected values. For example, the protein alpha-fetoprotein (AFP) is usually secreted by a pediatric liver tumor called hepatoblastoma. High levels of this protein can be used to aid in diagnosis, prognosis, and response to therapy.

Another example of a biochemical biomarker is urine catecholamines. These molecules are secreted by the sympathetic nervous systems and can be detected in the urine. High levels of these molecules aid in the diagnosis, response to therapy, and prediction of relapse in neuroblastoma. See Table 6-1 for biochemical biomarkers used in pediatric cancers and Table 6-2 lists the genetic biomarkers used in pediatric cancers.

Immunohistochemical staining

Immunohistochemical staining (IHC) testing uses antibodies to detect biomarkers, usually proteins, that are inside or on the surface of cancer cells in a tissue on microscope slides. The cancer tissue is taken via biopsy or surgical removal, prepared (either frozen or paraffin embedded) and stained using an antibody directed at the protein of interest. The pathologist then confirms the presence or absence of the protein under the microscope.

The protein PHOX2B, which was discussed above in the context of diagnostic biomarkers, is detected using IHC staining. Additional diagnostic biomarkers detected by IHC staining include myogenin for rhabdomyosarcoma, CD99 for Ewing sarcoma, CD19 for B acute lymphoblastic leukemia and others.

Genetic testing

Genetic testing is based on methods that measure changes in the DNA. The changes can be in the number or structure of chromosomes, genes and/or non-coding regions. The broader definition of genetic testing also encompasses changes in the number or structure of RNA molecules which are derived from DNA. Types of genetic tests can be broadly classified as sequencing-based methods and non-sequencing-based methods. Described below are the most common types of genetic testing encountered in pediatric cancer with examples for each type of test.

Non-sequencing based methods

Cancer cells usually change the size, number, and shape of chromosomes. The methods below differ not only by the way they detect chromosomal changes, but also but the minimal size of the change that can be detected.

Karyotype. This test produces an image of the cancer cells’ chromosomes and allows the detection of large chromosomal abnormalities. It requires growing the cancer cells in culture and typically takes 1 to 2 weeks to result.

Karyotyping is used as a prognostic biomarker in acute lymphoblastic leukemia. Specifically, patients with leukemia cells showing low number of chromosomes (less than 40 chromosomes on karyotype, also called hypodiploid) tend to be harder to treat than patients with high number of chromosomes (hyperdiploid).

Fluorescence in situ hybridization (FISH). This test uses a fluorescent probe that binds to specific regions of chromosomes and can detect regions where part of the chromosome is amplified, deleted, or fused to another chromosome (an aberration referred to as chromosomal translocation). This test can detect smaller changes than karyotype, is low cost and takes approximately 1 to 2 days for results. The disadvantage of this test is that it requires prior knowledge of the changed region in the DNA.

FISH is used to detect biomarkers in many types of childhood cancers including acute lymphoblastic leukemia, acute myeloid leukemia, neuroblastoma, Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, and others (See Table 6-2).

Single Nucleotide Polymorphism Microarray (SNP array). Occasionally, only small regions of the chromosome are amplified or lost and the orientation of the DNA letters can be inverted. Such changes cannot be detected using Karyotype or FISH. In such cases, a SNP array can help detect biomarkers by analyzing tumor DNA with hundreds of thousands of DNA polymorphisms or spelling changes in DNA that vary based on race and ethnicity. This allows for the detection of gains or losses of chromosomal regions without any prior knowledge of where these exist. SNP arrays are used to detect prognostic biomarkers in neuroblastoma and Wilms’ tumor.

Sequencing-based methods

Sequencing is the process of determining the sequence of letters of a DNA or RNA molecule. DNA and RNA sequencing technologies continue to evolve to allow for faster, cheaper, and more accurate results.

The traditional sequencing method referred to as Sanger sequencing or First Generation Sequencing is clinically used to determine the sequence of a single gene. First Generation Sequencing is accurate but time consuming. Next Generation Sequencing (NGS) is a technology that allows the sequencing of many genes or all of the gene in parallel. The following are sequencing-based genetic tests and their use in pediatric cancer.

Single gene test. This test is used to detect a change in a gene that is known to be modified in a type of cancer and for which a targeted therapy exists. For example, the gene ALK is mutated in approximately 15-25% of neuroblastomas. The presence of an ALK mutation may warrant the addition of an ALK-inhibitor to a treatment, and this is currently be tested in clinical trials. With the evolution of NGS, single gene testing is largely being replaced by NGS multi-gent strategies.

Targeted genetic panel. This test relies on NGS technology and is performed on a set of genes that are known to contribute to a specific type of cancer of a group of cancers. The number of genes included in a panel varies depending on the type of cancer and laboratory that performs the test, but typically is in the range of several hundred.

Germline testing. Most cancers are not inherited. However, well described cancer predisposition syndromes exist and offer an opportunity for biomarker testing. A liquid biopsy is a sequenced-based genetic testing that is performed on normal tissue, most often on blood or saliva, to detect a change in a gene or other regions in the DNA that affect all the cells of the body and could give rise to cancer.

Your oncologist may decide to order germline testing if other family members had cancer at a young age, if the cancer originates on both sides of the body (for example, in both eyes or both kidneys), and/or if your child has concerning features on physical exam. With the increased use of NGS on tumor tissue, it is now not uncommon to uncover a potentially concerning genetic predisposition mutation even if no family history exists. This typically leads to a referral to a genetic counselor for a discussion of the test result, potential confirmation testing, and further counseling as to what this means for the patient and family members.