Obtaining sufficient tissue and practicing proper specimen handling (which begins even before the specimen arrives in the pathology laboratory) are essential components for accurate pathologic diagnoses. The following section reviews the various types of biopsies, including liquid biopsies, used to sample tumors and important aspects of tissue fixation.
Although many types of tests may be used to make assessments that are suggestive of cancer, only a biopsy can be used to confirm a cancer diagnosis.
Methods for obtaining a tumor specimen
A biopsy is the removal of a small amount of tissue for pathology assessment. The goal of tissue biopsy is to obtain diagnostic tissue while minimizing morbidity, limiting potential tumor spread and avoiding interference with future treatments.
- If the tumor is palpable near the surface, the needle is guided by palpation.
- If the tumor is deeper in the body, then the needle is guided by imaging (typically ultrasound or CT scan).
Fine needle aspiration (FNA)
- Uses a thin (typically 22 gauge or smaller), hollow needle attached to a syringe. The needle used is even smaller than the ones used for blood tests.
- Removes individual cells, extremely small pieces of tumor tissue or fluid for pathologic evaluation.
- Multiple FNA passes can be made of a single large tumor in order to sample different sections.
- In cellular samples, material can be concentrated into a cell block for ancillary studies such as immunohistochemistry.
- Advantages: simple, rapid, minimally-invasive method that can be performed as an outpatient procedure. It is well tolerated with low risk for complications.
- Limitations: sampling errors due to low cellularity or inadequate tumor sampling.
- A pathologist may be present during image guided FNA procedures. If this is the case, the FNA aspirate is immediately processed on a slide and stained to preview the cells to ensure an adequate specimen was obtained. This is termed rapid on-site adequacy assessment.
Core needle biopsy
Source: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
- Uses a hollow needle that is slightly larger than the one used in FNA.
- Removes a small cylinder of tissue (about 1/16 inch in diameter and about 1/2 inch in length).
- Less invasive than surgery, but often requires local anesthesia.
- Advantages: In most cases, more tissue is obtained as compared to FNA, allowing more detailed ancillary studies to be performed. Histologic architecture is preserved as compared to FNA.
- Limitations: Limited sampling and inaccessibility of some masses (secondary to size, depth, density or location).
Source: National Cancer Institute, Linda Bartlett (Photographer)
- Either local or general anesthesia is required.
- More invasive than needle biopsies.
- Recovery time is required, increased morbidity and cost as compared to needle biopsy.
- A portion of a large tumor is removed.
- Typically only performed if the tumor is too large or too invasive to be removed in its entirety, or attempts at needle biopsy were non-diagnostic.
- Excisional biopsy - The entire tumor or suspicious area is removed.
- The entire tumor or suspicious area is removed.
- Typically some of the surrounding normal tissue is removed as well (termed the surgical margin).
- If an excisional biopsy specimen is found to be cancerous, the pathologist will examine the surgical margin to ensure that the tumor was removed in its entirety. This is determined based on whether there is a wide enough rim of normal tissue around the tumor.
Other types of biopsies
Scrape or brush cytology
- A small spatula or brush is used to scrape cells from the tissue being tested.
- Most common example is a Pap test.
- Other tissues commonly sampled in this way include the esophagus, the stomach, the bronchi, and the mouth.
- An endoscope is a thin, flexible, lighted tube that has a lens or camera on the end.
- Forceps may also be attached to the end of the tube and used to remove a small tissue sample of a suspicious area identified via the camera.
- An endoscope is used to visualize and biopsy different parts of the body, including the nose, sinuses, throat, esophagus, stomach and upper intestine.
- Some endoscopes are called a different name when they are used on a particular anatomic area. A bronchoscope is used to visualize and biopsy the lungs and bronchi. A colonoscope is used to visualize and biopsy the colon and rectum. A laparascope is used to visualize and biopsy the interior of the abdomen.
Bone marrow aspiration and biopsy
- Used to diagnose hematologic cancers including lymphoma, leukemia and multiple myeloma.
- Typically performed at the same time to examine the bone marrow.
- Bone marrow aspiration is used to sample a small amount of the liquid component of bone marrow. Bone marrow biopsy is used to remove a small amount of the solid tissue component of bone marrow.
- A wide needle is pushed into the bone. A sample of the liquid portion is removed using a syringe attached to the needle. The needle is then rotated to remove a sample of the bone.
- Most frequently performed on the pelvic bone.
Sentinel lymph node mapping and biopsy
- Termed sentinel lymph nodes because they “stand watch” over the tumor. They are lymph nodes that drain lymph fluid from the tumor tissue. A sentinel lymph node is defined as the first node or group of nodes to which cancer cells are most likely to spread from a primary tumor. Sentinel lymph node biopsy is most commonly used to help stage breast cancer and malignant melanoma, but it has been used for a variety of cancer types.
- Mapping involves using a colored dye and/or a radioactive material to trace the routes of lymph drainage from the tumor to identify the sentinel node(s).
- The sentinel node(s) are then removed and examined microscopically to determine if they contain cancerous cells. A negative sentinel lymph node biopsy result suggests that the cancer has not spread to regional lymph nodes or other organs. If the sentinel lymph node(s) are negative, then no additional regional lymph nodes are removed at surgery because the tumor has not yet metastasized to the lymph nodes. If cancerous cells are found, then the remaining lymph nodes in the area may be removed in a process termed lymph node dissection.
Liquid biopsy technology is a rapidly emerging field. The terminology liquid biopsy came about because we are rapidly moving to an era where some of the traditional assessments done with a tissue biopsy (e.g., molecular marker testing) can now be done in blood, urine or other bodily fluid that is less invasive than a tissue biopsy. Currently, about 40 companies have developed assays to detect cell-free circulating DNA (cfDNA), circulating tumor DNA (ctDNA), or circulating tumor cells (CTCs).
Tumors shed cells (CTCs) into the bloodstream, which can be isolated for analysis. The challenge is that there are very few tumor cells, but there are a number of advantages in isolating them for analysis. The most obvious advantage is that the patient would not need to undergo an invasive biopsy procedure and only would have a blood sample drawn. This permits easier serial analysis over time to monitor a tumor’s changes to better guide therapy changes as the tumor progresses. Additionally, tumors are heterogeneous, making it challenging to obtain a molecular representation of the tumor from a small biopsy sample (such as those from fine needle aspiration or core needle biopsy). Treating a patient with a tumor based on the analysis of a small biopsy may result in only a portion of the cells being effectively targeted. Using CTCs, the heterogeneity of a tumor is better represented, as the cells can come from multiple locations within a tumor as well as from multiple tumors in the case of metastatic disease. Research has shown that the number of CTCs reflects the state of disease — having more CTCs corresponds with more disease. Circulating tumor cells that have been isolated can also be sequenced individually or as a group to identify actionable targets for treatment. Several companies, e.g, CellSearch, Biocept, etc., have commercialized CTC analysis. A challenge in utilizing CTCs for diagnostics is the low numbers found in the blood at any time. While the numbers increase with metastatic state, they are still few compared to the number of red and white blood cells. Companies have developed proprietary collection methods to stabilize the CTCS in the samples sent to them overnight for isolation upon arrival in the lab. Further research is needed to improve this approach.
Dying cells release DNA into the bloodstream (cfDNA). Tumor cells do this as well (ctDNA), but typically, only a small portion of the total DNA is found in the blood. The amount of tumor DNA found in the blood typically correlates with stage, increasing with stage and number of metastases. Multiple approaches are used by different companies to analyze the ctDNA molecularly, but the most common utilize polymerase chain reaction or next-generation sequencing to identify molecular alterations in the DNA. The same advantages of isolating CTCs for analysis vs. biopsies apply to ctDNA, but it is often possible to obtain higher quantities of ctDNA than CTCs.
Currently, a few companies are expanding the concept of liquid biopsies, by using other bodily fluids, such as urine and cerebral spinal fluid (for brain tumors and metastases). Urine provides an interesting option if proven successful because the sample can be collected at home and shipped by the patient, providing the most convenient and least invasive option of all. For more information, see the section "Liquid Biopsies."
Wafik El-Deiry MD, discusses the pros and cons of both tissue and liquid biopsies.
Comparison of tissue and liquid biopsies
Abbreviations: CTC, circulating tumor cell; ctDNA, circulating tumor DNA; EGFR, epidermal growth factor receptor.
Tissue fixation serves several purposes during the pathologic evaluation of specimens. Fixation preserves tissue by preventing autolysis by cellular enzymes, helps prevent decomposition of tissue by bacteria and molds, hardens tissue to facilitate sectioning, inactivates infectious agents and enhances tissue avidity for dyes. Fixation also has undesirable effects on tissue such as alteration of protein structure (loss of antigenicity), loss of soluble tissue components and degradation of DNA and RNA.
Types of fixatives
- Formalin: The standard fixative used in the pathology laboratory is 10% phosphate-buffered formalin. It fixes most tissues well and is compatible with most ancillary testing such as immunohistochemistry and molecular tests.
- Bouin solution (picric acid, formaldehyde and acetic acid): Fixation in Bouin solution results in sharp hematoxylin and eosin staining and is preferred by some pathologists. Disadvantages include decreased sensitivity of immunohistochemical tests and increased degradation of DNA and RNA by picric acid.
- B5 (mercuric chloride, sodium acetate and formalin): B5 is often used for routine fixation of lymph nodes, spleens and other tissue if a lymphoproliferative process is suspected. B5 provides rapid fixation with excellent cytologic details and antigen preservation for lymphoid markers. Tissue may become brittle if over-fixation occurs with B5, and special procedures for disposal are needed due to the presence of mercury.
- Glutaraldehyde: The fixative glutaraldehyde is used for tissue that is to be evaluated by electron microscopy.
Creating formalin-fixed paraffin-embedded tissue blocks
After appropriate fixation, tissue, in blocks, is placed into a processor that dehydrates tissue through a series of graded alcohol baths and infiltrates the tissue with paraffin wax, resulting in a formalin-fixed paraffin-embedded tissue block. Tissue from these blocks is then sectioned thinly (0.4 µm to 0.5 µm) using a microtome and placed onto a glass slide. Tissue on the slides is stained with hematoxylin and eosin and covered with a coverslip before examination by a pathologist.
Effect of time on fixation
Several factors related to tissue fixation may affect the results of ancillary studies such as immunohistochemical and molecular testing. Autolysis begins immediately after tissue is removed from a patient. Although autolysis can be reduced by refrigeration, delays before fixation can adversely affect the diagnostic quality of tissue. The time between when a specimen is removed from a patient to when it is in contact with formalin is called the cold ischemic time. Extended cold ischemic times (greater than 1 hour) may result in false-negative testing for markers such as estrogen receptor, progesterone receptor and HER-2. It is important that specimens are transported to the lab in a timely fashion to avoid extended cold ischemic times. An adequate amount of fixative in the specimen container is usually considered to be 15 to 20 times the volume of the tissue. However, even in this short time, changes in phosphorylation of important proteins occur (both increase and decrease at specific sites has been noted).
The process of fixation is a chemical reaction that usually requires a minimum of 6 hours (even for small biopsy specimens) to reach sufficient tissue fixation. Certain tissue types, such as those containing a high content of adipose tissue, and larger specimens require longer fixation times. Larger tissues from resections may also require opening up to enable the fixative to enter all areas and provide even fixation. Both under-fixation and over-fixation of tissue may result in loss of antigenicity and degradation of RNA and DNA. Specific ASCO/College of American Pathologists guidelines for fixation of breast specimens exist to preserve antigenicity of tissue for hormone receptor and HER-2 testing. Breast specimens are to be fixed for a minimum of 6 hours and no more than 72 hours in 10% neutral buffered formalin. These guidelines may be applied to other specimen types in an attempt to standardize pre-analytical variables for ancillary testing.
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