Laboratory Tests in Childhood Cancer

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A patient’s medical team uses the specific, detailed information from laboratory testing to create a treatment plan for that individual case.

Blood cancers such as leukemia are diagnosed by examining blood and bone marrow. Other types of cancer are typically diagnosed by examining tissue removed from a suspected tumor during a biopsy. The sample of tissue removed for testing is called a specimen.

A pathologist reviews an enlarged view of tissue on a large format monitor. — Pathologists specialize in different areas. It is important that specimens are examined by pathologists who have specialized training for the specific cancer type they are evaluating.

These tests take place in a pathology lab, led by a pathologist, a medical doctor who specializes in diagnosing disease by studying cells, tissues, and body fluids. Like other medical doctors, pathologists specialize in different areas.

Hematopathologists focus on diagnosing cancers of the blood, such as leukemias and lymphomas.
Anatomic pathologists study body organs and tissues. In cancer diagnosis, anatomic pathologists focus on types of tumors such as sarcomas and carcinomas.
Neuropathologists are a special type of anatomic pathologist that focus on diagnosing cancers of the brain and spinal cord.

It is important that specimens are examined by pathologists who have specialized training for the specific cancer type they are evaluating.

How specimens are collected

Cancers of the blood

In cases of suspected cancer of the blood, a sample of blood or bone marrow (through a bone marrow aspiration and/or biopsy) will be collected from the patient. The staff labels the container with the patient’s information, and the specimen is sent to a pathology laboratory.

Tumors of solid tissue

When a patient has a suspected tumor in the brain or other parts of the body, all or part of it may be removed during surgery. When only a portion of the tumor is removed and evaluated, this is called a surgical biopsy. In other surgical procedures, surgeons remove as much of the tumor as possible. This is referred to as a resection.

The removed tissue must be cut into thin sections, placed on slides, and stained with dyes before it can be examined under a microscope. Two methods are used to make the tissue firm enough to cut into thin sections:

Frozen section - Tissue is rapidly frozen.
Permanent section - Tissue is embedded in a special wax called paraffin.

All tissue samples are prepared as permanent sections, but sometimes frozen sections are also prepared.

In surgical biopsies, the staff may prepare a frozen section so a pathologist can analyze the biopsy specimen during the surgery. The specimen is frozen quickly and prepared so a pathologist can examine it in a laboratory close to the operating suite. The quality is not as high as with a permanent section, but the pathologist can usually quickly determine if the tissue is cancerous. This knowledge helps surgeons make immediate decisions about surgery.

In some cases of suspected tumors of solid tissue, another type of biopsy – a needle biopsy – is performed. In a needle biopsy, a sample of tissue is removed with a needle. A member of the team who performed the biopsy will place the specimen in a sterile container and a special liquid will be added to preserve it. It will be labeled with the patient’s information and sent to the pathology laboratory for examination and testing.

What happens in the laboratory

The pathology lab staff prepares the sample and performs different types of tests to gather information about the tissue. The pathologist includes this information in a report for the oncologist. The pathologist and oncologist work together to make a diagnosis.

Gross examination

First, a pathologist will examine the specimen and describe its appearance to the naked eye. This is called a gross examination. This description will include the specimen’s color, size, and other features.

Preparation of specimen

The laboratory staff then prepares the specimen for the pathologist to look at under a microscope. The specimen is processed in a machine that places the tissue in a paraffin wax block. The staff cuts the block of tissue into thin slices to mount on slides (tissue sections). Slides are small, flat pieces of glass used to view objects under a microscope.

After this process, a technician will stain the tissue section with dyes (typically with hematoxylin and eosin) which help highlight different features of the tissue under the microscope. The nucleus of each cell will appear blue. The rest of the cell (cytoplasm) will appear pink.

Sample histology slide shows tissues stained so that the nucleus of the cells appear blue and the rest of the cell appears pink. — Sample histology slide shows tissue stained with hematoxylin and eosin to highlight different features of the tissue under the microscope.

Microscopic examination

The pathologist looks at the tissue sections under a microscope to see how they compare with normal cells. Viewing the specimen in this manner is called histology. Histology is the study of structures of cells and tissue.

The pathologist creates the report based on what he or she sees under the microscope and the results of other tests performed on the tissue. It is written in technical, medical language that may be hard to understand. Ask your care team if you have any questions.

In general, the pathologist describes the specimen’s features:

The types of cells
How the cells are arranged
Whether the cells are cancerous
Other features helpful in diagnosis and treatment such as whether the tumor cells demonstrate aggressive features (often referred to as tumor grade) or if the tumor cells have spread into other normal tissues (often referred to as the tumor stage). In some circumstances, pathologists also determine if the tumor has been completely removed from the patient or if the edge of the specimen (margins) are positive for cancer.

In addition to evaluation under the microscope, the specimen may undergo further tests and analysis. These results will also be included in the pathology report.

Types of laboratory tests

Immunohistochemistry sample shows blasts that have both T-cell markers in red and megakaryocyte markers in brown. — Immunohistochemistry sample

Immunophenotyping

Immunophenotyping is a process that uses antibodies to identify cells based on the types of proteins (or markers) on the surface of the cells. This process is used to diagnose specific types of cancer by comparing the protein antigens on cancer cells to those found in normal cells. Immunophenotyping includes immunohistochemical staining and flow cytometry.

Immunohistochemical staining

Immunohistochemistry is a process that uses antibodies to determine if specific proteins are present in a sample of tissue. This type of staining helps pathologists recognize abnormal cells under the microscope and can help pathologists establish a diagnosis or identify relapse.

Flow cytometry

Flow cytometry is a method of measuring the number of cells in a sample, the percentage of live cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface. Antibodies are tagged (linked) with a light-sensitive dye, placed in a fluid, and passed in a stream before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts to the light. The process is extremely sensitive. It can look at many thousands of cells per second and identify even 1 leukemia cell among thousands of blood cells. Flow cytometry can detect cancer cells that would not be visible under a microscope. This is known as minimal residual disease, which is important in leukemia treatment.

Molecular and genetic tests

The results of molecular and genetic tests help guide treatment for certain cancers. These types of tests may look at chromosomes, genes, DNA, RNA, and proteins.
Chromosomes contain genes. Genes are segments of DNA that contain the code for specific proteins. Proteins carry out specific functions of a cell. They are the basis of body structure and needed for the body to function properly.
Molecular and genetic testing can often tell a physician whether certain chemotherapy drugs may or may not work. Changes in genes called mutations may result in cancer. In some cancers, a mutation may lead to an increased amount of a particular protein in a tumor tissue or to production of a protein that has abnormal activity. Tumors with certain mutations may be more aggressive and/or more resistant to chemotherapy. Sometimes a certain mutation may mean a tumor will be more vulnerable to certain drugs.

Cytogenetics

Cytogenetics is the study of chromosomes. Chromosomes are long strands of DNA. DNA are molecules inside cells that contain genetic information that is passed from one generation to the next.

Cytogenetics involves testing samples of tissue, blood, or bone marrow in a laboratory to look for changes in chromosomes, including broken, missing, or extra chromosomes. Changes in certain chromosomes may be a sign of cancer or a genetic disease or condition. Cytogenetics may be used to help diagnose a disease, plan treatment, and find out how well treatment is working.

Sample FISH images include an example of the MLL gene rearrangement. The FISH probe is at the gene location and shows normal if yellow. If the gene is split, the yellow separates into green and red signals. This example shows a separation that is sometimes seen in patients with acute myeloid leukemia. — These FISH images show an example of the MLL gene rearrangement in a pediatric acute myeloid leukemia patient. The FISH probe is at the gene location and shows normal if yellow. If the gene is split, the yellow separates into green and red signals.

FISH (fluorescence in situ hybridization)

FISH is a laboratory technique that can detect and locate a specific DNA sequence on a chromosome. Pieces of DNA that contain a fluorescent dye are made in the laboratory and added to cells or tissues on a glass slide. When these pieces of DNA bind to specific genes or areas of chromosomes on the slide, they light up when viewed under a microscope with a special light. FISH can detect abnormalities that cannot be found with standard analysis of chromosomes.

Pathologists are looking for certain abnormalities that drive cancer growth:

Too many copies of certain oncogenes, genes that lead to the growth of cancer cells
Deleted copies of tumor suppressor genes, genes that contain instructions to prevent the growth of cancer
Rearrangements – A part of a gene breaks off and attaches to another gene where it does not belong. It creates a fusion protein that causes the growth of cancer cells.

PCR (polymerase chain reaction)

PCR is a laboratory method used to make many copies of a specific piece of DNA from a sample that contains very tiny amounts of that DNA. It is sometimes called molecular “photocopying.” PCR allows these pieces of DNA to be amplified (enlarged) so they can be detected.

PCR may be used to look for certain changes in a gene or chromosome, which may help find and diagnose a cancer. It may also be used to look at pieces of the DNA of certain bacteria, viruses, or other microorganisms to help diagnose an infection.

Because large amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. PCR can produce a billion copies of sequences in just a few hours.

PCR allows scientists to identify mutations of genes and confirm the presence of fusion proteins that cause cancer growth.

DNA sequencing

DNA sequencing is a laboratory process used to learn the exact sequence (order) of the four building blocks, or bases, (identified by the letters A, C, G, and T) that make up DNA. DNA sequencing can be used to find DNA mutations (changes) that may cause cancer.

DNA sequencing tests can have a wide or targeted focus. Targeted DNA sequencing tests, also called multigene panels, analyze specific mutations. Some targeted sequencing tests analyze alterations that are common in a single cancer type. Others analyze alterations that may be found in many cancer types.

Broad DNA sequencing tests analyze the sequence of large regions of DNA rather than specific mutations. These tests include whole exome sequencing and whole genome sequencing.

Whole genome sequencing reads the sequence of all of the DNA in a patient’s cells – known as the genome. Whole exome sequencing reads the sequence of all of a patient’s genes, known as the exome. Most cancer-causing DNA changes occur in genes, but DNA changes outside of genes can also drive cancer growth.

Finding out the results

Sometimes all testing and analysis happen at the medical center where the biopsy was performed. Other times, the specimen must be sent to laboratories located somewhere else. Some tests require a series of steps and procedures and may take a few weeks for results.

The pathologist will include information from these tests into the pathology report. It is shared with the oncologist. The oncologist will share results of the pathology report during a clinic visit.

The more information doctors have about the cancer the better they can develop the most effective therapies for that individual case of cancer.

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Reviewed: July 2018