Much of the testing process takes place "behind the scenes." You will have a chance to meet and ask questions of most of your health care team, which may include a surgeon, medical oncologist, radiation oncologist, oncology nurses, and others. You will be able to see at least parts of what these professionals do. On the other hand, you rarely meet the pathologists, histotechnologists, cytotechnologists, and medical laboratory technologists whose work can tell you whether your biopsy is malignant (cancerous) or benign (non-cancerous).

This document follows a tissue or cell sample as it goes through the steps of the diagnostic process, starting when the doctor removes the sample. It then explains what is done to the sample in the laboratory during the testing process, including some new ways that the sample may be tested. Finally, this document explains what type of information the pathology report contains. Along the way, it explains how the pathology information affects treatment decisions, and defines some of the medical terms used in these reports.

How Is Cancer Diagnosed?

A diagnosis of cancer is nearly always based on an expert looking at cell or tissue samples under a microscope. The procedure that takes a sample for this testing is called a biopsy, and the tissue sample is called the biopsy specimen. The testing process is sometimes referred to as pathology.

Lumps that might be malignant (cancerous) can be found by imaging (radiology) studies or felt as masses (lumps) during a physical exam, but they still must be sampled and looked at under a microscope to find out what they are. Not all lumps are malignant. In fact, most tumors are benign (not cancer). A cancerous tumor is able to spread into surrounding tissues and even to distant parts of the body. A benign tumor can not do this.

Types of Tissue and Cell Samples

Tissue or cell samples can be removed from almost any part of the body. How this is done depends on where the tumor is and what type of cancer is suspected. For instance, the methods used for skin biopsies clearly need to be different from the procedures for brain biopsies.

Overview of Biopsy Types

Some types of biopsies involve operations to remove an entire organ. These types are done only by surgeons. Other types of biopsies are less invasive and may remove tumor samples through a thin needle or through an endoscope (a flexible lighted tube). These biopsies are often done by surgeons, but can also be done by other doctors. The most common biopsy types used in cancer diagnosis are discussed in this section. For more complete information, refer to the diagnosis section of American Cancer Society documents on specific types of cancer.

Needle Biopsy

There are 2 types of needle biopsies:

• fine needle biopsy (also called fine needle aspiration) 
• core needle biopsy (also called core biopsy)

Fine needle aspiration (FNA) uses a very thin needle and a syringe to withdraw a small amount of fluid and very small pieces of tissue from the tumor mass. The doctor can aim the needle while feeling a suspicious tumor or area near the surface of the body. If the tumor is deep inside the body and cannot be felt, the needle can be guided while being watched by imaging procedures such as an ultrasound or a computed tomography (CT) scan. The main advantages of FNA are that it does not require an incision (cutting through the skin) and that in some cases it is possible to make a diagnosis the same day. The disadvantage is that sometimes this needle cannot remove enough tissue for a definite diagnosis. Although FNA is a type of biopsy, it is also classified as a cytology test (see next section).

The needles used for a core biopsy are slightly larger than those used in FNA. They remove a small cylinder of tissue (about 1/16 inch in diameter and 1/2 inch long). The core needle biopsy is done using local anesthesia (numbing medicine) in the doctor's office or clinic. Like FNA, a core biopsy can sample tumors that can be felt by the doctor as well as smaller ones that must be seen using imaging studies. Doctors sometimes use special vacuum tools to get larger core biopsies from breast tissue. (For more information, see the American Cancer Society document, For Women Facing a Breast Biopsy.) Processing core biopsy samples usually takes longer than processing FNA biopsies.

Excisional or Incisional Biopsy

With this type of biopsy, a surgeon cuts through the skin to remove the entire tumor (excisional biopsy) or a small part of a large tumor (incisional biopsy). This often can be done using local anesthesia or regional anesthesia (numbing medicine). If the tumor is inside the chest or abdomen, general anesthesia is used (the patient is asleep).

Endoscopic Biopsy

This is done using a thin, flexible lighted tube that has a lens or a video camera. If a video camera is used, it is connected to a screen that allows the doctor to clearly see any masses in the area. The endoscope can be passed through a body opening to look at suspicious areas in the swallowing tube (esophagus), stomach, intestine, urine tube (urethra), bladder, uterus (womb), or breathing tubes (bronchi). Advantages of endoscopy include the chance to see the cancer directly and the ability to take a small tissue sample through the endoscope to find out if cancer is present and, if so, the cell type.

Laparoscopy, Thoracoscopy, and Mediastinoscopy

Laparoscopy is similar to endoscopy but is used to look inside the abdomen and remove tissue samples. A small incision is made in the abdomen then the endoscope is passed through this opening to see the inside. Similar procedures to look inside the chest are called thoracoscopy and mediastinoscopy.

Laparotomy and Thoracotomy

A laparotomy is a type of surgery that involves an incision into the abdomen, usually a vertical incision from upper to lower abdomen. This may be done when there is uncertainty about a suspicious area that cannot be diagnosed by less invasive tests. During the laparotomy, a biopsy of a suspicious area can be taken and the doctor can look at its location and size. The areas nearby can be checked to see if they are involved. General anesthesia is used for this technique. A similar operation which opens the chest is called thoracotomy.

Skin Biopsies

There are several ways to take a biopsy of the skin. Doctors choose the one best suited to the type of skin tumor suspected. Shave biopsies remove the outer layers of skin and are fine for some basal cell or squamous cell skin cancers, but they are not recommended for suspected melanomas of the skin. Punch biopsies or excisional biopsies remove deeper layers of the skin, and can find out how deeply a melanoma has gone into the skin -- an important factor in choosing treatment for that type of cancer.

Sentinel Lymph Node Mapping and Biopsy

This is a way for the surgeon to choose which lymph nodes to remove for an excisional biopsy. Sentinel node mapping and biopsy has become a common way to find out whether the cancer has spread to the lymph nodes (especially melanoma and breast cancer). This procedure can find the lymph nodes that drain lymph fluid from the area where the cancer started. If the cancer has spread, these lymph nodes are usually the first place it will go. That is why these lymph nodes are called "sentinel" nodes (meaning that they stand watch over the tumor area, so to speak).

To find the sentinel lymph node (or nodes), the doctor injects a small amount of slightly radioactive material into the area of the cancer. By checking various lymph node areas with a machine that detects radioactivity (like a Geiger counter), the doctor can find the group of lymph nodes the cancer is most likely to travel to. Then the doctor injects a small amount of a harmless blue dye into the site of the cancer. After about an hour, a surgeon makes a small incision in the lymph node area that was found with the radioactive test. Those lymph nodes are then checked to find which one(s) turned blue or became radioactive. When the sentinel node has been found, it is removed (an excisional biopsy) and looked at under a microscope. If the sentinel node does not contain cancer cells, no more lymph node surgery is needed because it is very unlikely the cancer would have spread beyond this point. If cancer cells are found in the sentinel node, the rest of the lymph nodes in this area are removed and looked at as well. This is known as a lymph node dissection.

Overview of Cytology Types

Diagnosing diseases based on looking at single cells and small clusters of cells is called cytology or cytopathology. It has become more important in cancer diagnosis over the past few decades. While the pieces of tissue in biopsy samples may be as small as 1/16 inch or much larger (several inches), the individual cells and the cell clusters in cytology samples are usually too small to see without a microscope. Sometimes, as in some FNA samples, only one drop of blood or tissue fluid (containing tiny fragments of the tumor) is taken. On the other hand, some pleural fluid (from around the lung) or peritoneal fluid (from inside the abdomen) cytology samples may include a quart or more of fluid.

A cytology specimen usually:

• is easier to get 
• causes less discomfort to the patient 
• is less likely to result in serious complications 
• costs less than a tissue biopsy

The disadvantage is that, in some cases, a tissue biopsy result is more accurate, but in many cases, the cytology fluid may be just as accurate. Sometimes an excisional biopsy is the only treatment needed to remove a cancer -- which is an obvious advantage. In other cases, a cancer might be better treated by chemotherapy or radiation therapy, and surgery might be done after these treatments. For those types of cancer, a cytology sample, endoscopic biopsy, or incisional biopsy might be a better choice. As you can see, choices of tests are not simple -- the doctors consider many factors about the specific type of cancer that is suspected and what organ is affected.

Cytology tests may be used in two ways -- for diagnosis or for screening.

A diagnostic test is only used for people who have signs, symptoms, or some other reason to suspect that a particular disease such as cancer is likely to be present. A diagnostic test finds out if a disease is present and, if so, it precisely and accurately classifies the disease.

A screening test is used to find people who might have a certain disease even before they develop symptoms. A screening test is expected to find nearly all people who are likely to have the disease, but the screening test does not prove that the disease is present. That means a diagnostic test is used if a screening test is positive (that is, if something is found on the screening test). Some cytology tests, such as the Pap test, are mainly used for screening but in many cases can accurately identify cancers (see "Scrape or Brush Cytology" below). A biopsy is generally done to be sure of any abnormal finding before surgery or radiation is started.

Fine Needle Aspiration (FNA) Biopsy

FNA is sometimes considered a cytology test and is sometimes called a biopsy. It is discussed in this document in the previous section, "Overview of Biopsy Types."

Body Fluids

This term refers to fluid from cavities and spaces in the body. These fluids can be tested to see if cancer cells are present. Some of the body cavity fluids tested in this way include:

• urine 
• sputum (phlegm) 
• spinal fluid, also known as cerebrospinal fluid or CSF (from the space surrounding the brain and spinal cord) 
• pleural fluid (from the space around the lungs) 
• pericardial fluid (from the sac that surrounds the heart) 
• ascitic fluid, also known as ascites or peritoneal fluid (from the space in the abdomen or chest)

Scrape or Brush Cytology

Another cytology technique is to gently scrape or brush some cells from the organ or tissue being tested. The best-known cytology test that samples cells in this way is the Pap test. Pap test samples are taken by using a small spatula and/or brush to remove cells from the cervix (lower part of the uterus or womb). Other areas that can be brushed or scraped include the esophagus, stomach, bronchi (breathing tubes that lead to the lungs), and mouth.

What happens to biopsy and cytology specimens after they are removed from the patient?

Routine Biopsy Processing for Histology

There are standard procedures and methods that are used with nearly all types of biopsy samples. These procedures are the usual ways that a sample is prepared for use by the doctor. Additional procedures, which are described later in this document, may also be done on certain types of samples (such as lymph nodes and bone marrow).

After the doctor gets the biopsy specimen, it is placed in a container with formalin (a mixture of water and formaldehyde) or another fluid to preserve it. The container is labeled with the patient's name and other identifying information (hospital number and birth date, for example), site of biopsy (exactly where on the body it was taken from), and then sent to the pathology lab with a paper called a pathology requisition form. This form also identifies who submitted the biopsy, the date the biopsy was obtained, and certain clinical history (information about the patient's symptoms, other abnormal test results, and what type of disease the doctor expects the biopsy may show). Next the pathologist or an assistant looks at the specimen without a microscope. How the whole sample looks before further processing is called the "gross description" and includes the tissue sample's size, color, consistency, and other characteristics. The lab staff may even take a picture of the sample as part of the record.

The gross examination is important since the pathologist often sees features that suggest cancer. This will help the pathologist decide which parts of a large biopsy are the most critical to study under a microscope. For small biopsies, for example, like a punch biopsy or a core needle biopsy, the entire specimen may be looked at under a microscope. The tissue to be looked at under the microscope is placed into small containers called cassettes. The cassettes hold the tissue securely while it is processed, and help keep small samples from getting lost. After processing, which may take a few hours but is usually done overnight, the tissue sample is placed into a mold with hot paraffin wax. The wax cools to form a solid block that protects the tissue. This paraffin wax block with the embedded tissue is placed on an instrument called a microtome, which the histotechnologist uses to cut very thin slices of the tissue. These thin slices of the specimen are placed on glass slides, and dipped into a series of stains or dyes to change the color of the tissue. The color makes cells more distinctive when viewed under a microscope. For most biopsy specimens, routine processing as described above is all that is required. At this point (usually the day after the biopsy was performed), the pathologist looks at the tissue under a microscope. Looking at the solid specimens in this way is called histology, which is the study of the structures of cells and tissues.

Intra-operative Consultation (Frozen Section)

Sometimes a surgeon needs information about a tissue sample during surgery, so that decisions can be made about immediate surgical treatment. The surgeon cannot wait until the next day as is the case for routine biopsies. He or she will request an intra-operative (during surgery) pathology consultation. This consultation is often called a frozen section exam.

When a frozen section exam is performed, fresh tissue is sent from the operating room directly to the pathologist. Because the patient is often under general anesthesia it is important that the tissue be looked at as quickly as possible. This usually takes 10 to 20 minutes. The fresh tissue is grossly examined by the pathologist to decide which part of the tissue sample should be looked at under the microscope. Instead of processing the tissue in wax blocks, the tissue is quickly frozen in a special solution that forms what looks like an ice cube around the tissue sample. It is then thinly sectioned (sliced) on a refrigerated microtome, quickly stained (dipped in a series of dyes), and .looked at under the microscope. Although the frozen sections usually do not display features of the tissue as clearly as sections of tissue embedded in wax, they are usually provide enough information to help the surgeon decide what type of operation, if any, is best for the patient.

Frozen sections are often used to evaluate how completely a cancer has been removed. For example, if a lobe of a lung is removed due to cancer, the surgeon will want to know whether the bronchial (breathing tube) margin (the edge of the removed tissue) is cancer-free. A slice from the edge of the tissue that was removed is then sent for a frozen section diagnosis. If there is no cancer in that margin, additional surgery usually is not necessary. But if cancer cells are found, it is assumed that some cancer cells are still present in the tissue left in the patient. So the surgeon will usually remove more tissue, in order to try to get all the cancer cells and reduce the chance of cancer growing back. If it is not possible to remove more tissue, there may be other options such as radiation to destroy the remaining cancer cells.

Intra-operative consultations do not always provide a definite answer. In some cases, a piece of tissue will require routine or even special processing to arrive at a clear answer. In such cases, the surgeon will usually close the surgical incision. When the results are available in a few days, another operation may be needed.

Cytology Specimen Processing

Processing of cytology specimens depends greatly on their type. Some specimens are smeared directly on glass microscope slides by the doctor who obtains the sample. The slides, which are called smears, are then sent to the cytology lab where they are dipped into a series of stains (colored dyes), similar to those used for biopsy samples. Other specimens, such as body fluids, cannot be easily placed on a glass microscope slide because they are too dilute (there are too few cells in a large volume of fluid). Several methods are used in cytology labs to concentrate the cells on a glass slide before staining. After processing and staining, the samples are examined under a microscope by a cytotechnologist, who can locate abnormal cells and mark their location with a special pen. A pathologist will then review the marked cells and decide on a diagnosis.

What do doctors look for under the microscope?

General Principles

Over a hundred years ago, scientists realized that various tissues and organs look different from each other under a microscope. This is because they are formed by different cell types and because the cells are arranged differently. Even more importantly, it was discovered that the usual appearance of each type of tissue or organ is changed by certain diseases such as cancer. During the past century, this science, known as pathology, has been greatly refined.

Most tissue and cell samples are looked at by pathologists (doctors who specialize in diagnosing diseases by laboratory tests). Sometimes, other doctors will also examine specimens or tissues of organs related to their area of expertise. For example, hematologists often examine blood and bone marrow samples from their patients, and some dermatologists will examine their patients’ skin biopsy specimens.

The details of how doctors can tell normal tissue from cancer, and recognize the different types of cancer, are the subject of many thousands of pages of medical textbooks and journals. Some features that doctors look for under a microscope are important only when found in 1 or 2 types of tissue, while others will be more important if found in almost all tissues. There a few general concepts that can be explained in less technical terms and can help you to better understand how doctors decide whether cancer is present.

• The overall size and shape of cancer cells are often abnormal. They may be either smaller or larger than normal cells. Normal cells often have certain shapes that help them better perform their roles in the body. Cancer cells usually do not function in a useful way and their shapes are often distorted. Unlike normal cells that tend to have the same size and shape, cancer cells often are very different in their sizes and shapes. 
• The size and shape of the nucleus of a cancer cell is often abnormal. The nucleus is the center of the cell that contains the cell's DNA (deoxyribonucleic acid). The nucleus is surrounded by cytoplasm. Some types of cells can be imagined as looking like a fried egg, in which the central yolk represents the nucleus and the surrounding white is the cytoplasm (this is only a way of imagining cells, and does not truly reflect what cells are made of). Cancer cells typically have a nucleus that is larger than that of a normal cell. And, like the overall cell size and shape, the size and shape of the cell nucleus is generally similar among normal cells of each tissue but can vary greatly among cancer cells. Another feature of the nucleus of a cancer cell is that it appears darker when seen under a microscope after being stained with certain dyes. The nucleus from a cancer cell is larger and has a darker shade because it often contains too much DNA. 
• Cancer cells do not relate to each other normally. Normal tissues are formed by a very orderly arrangement of cells. The arrangement of normal cells reflects the function of each tissue. For instance, cells can form glands that produce substances that are taken to other parts of the tissue. Gland tissue in the breast is organized into lobules, which, during breast-feeding can produce milk, and ducts that carry milk from the lobules to the nipple. Cells of the stomach also form glands, to produce enzymes, acid, and mucus that digest the food and protect the stomach lining from digesting itself. When cancers develop in the breast, stomach, and many other tissues, the cancer cells do not form glands as they should. Sometimes the cancer cells form abnormal or distorted glands. Sometimes they form cell clumps that do not look like glands at all. Another feature that shows abnormal interactions by cancer cells is that cancer cells invade other tissues. Normal cells stay where they belong within a tissue. The ability of cancer cells to invade reflects the fact that their growth and movement is not coordinated with their neighboring cells. This ability to invade is how cancer spreads to and damages nearby tissues. And, unlike normal cells, cancer cells can metastasize (spread through blood vessels or lymph vessels) to distant parts of the body. Knowing this helps doctors recognize cancers under a microscope, because finding cells where they don't belong is a useful clue that they might be cancerous.

Classification of Various Types of Cancer

There are several basic kinds of cancers, which doctors can further classify into hundreds or even thousands of types, based on how they look under a microscope. Cancers are named according to which type of normal cells and tissues they most closely resemble. For example, cancers that look like glandular tissues are called adenocarcinomas. Other cancers that resemble certain immune system cells are called lymphomas, and those that look like bone or fat tissue are osteosarcomas and liposarcomas, respectively.

Grading a Cancer

In addition to identifying the cell type or tissue a cancer looks like, doctors decide how close that resemblance is -- the grade of the cancer. Cancers that look more like normal tissues are called low grade, and those that do not resemble normal tissues are high grade. A high-grade cancer tends to grow and spread more quickly than a low-grade cancer. Patients with high-grade cancers tend to have a poorer prognosis (outlook).

Special Studies in Cancer Diagnosis

Although the type and grade of a cancer is usually clear when it is seen under a microscope after routine processing and staining, this is not always the case. Sometimes the pathologist may need extra laboratory methods to make a diagnosis.

Histochemical Stains

These tests use a different chemical dyes that are attracted to certain substances found in some types of cancer cells. An example is the mucicarmine stain, which is attracted to mucus. Droplets of mucus inside a cell that are exposed to this stain will appear pink-red under a microscope. This stain is useful if the pathologist suspects, for example, an adenocarcinoma (a glandular type of cancer) in a lung biopsy. Adenocarcinomas can produce mucus, so finding pink-red spots in lung cancer cells will tell the pathologist that the diagnosis is adenocarcinoma.

Besides being helpful in sorting out different kinds of tumors, other types of special stains are used in the laboratory to identify microorganisms (germs) like bacteria and fungi in tissue sections. This is important to people with cancer that may develop infections as a side effect of chemotherapy, radiation, or even because of the cancer itself. It is also important in cancer diagnosis because some infectious diseases cause lumps to form which might be confused with a cancer at first, until these histochemical (cell-affecting) stains prove that the patient has an infection and not cancer.

Immunohistochemical Stains

Immunohistochemical or immunoperoxidase stains are another very useful category of special tests. The basic principle of this method is that an antibody will attach itself to certain substances called antigens. Each type of antibody recognizes and attaches to antigens that fit it exactly. Certain types of normal cells and cancer cells contain unique antigens, which can be recognized by specific antibodies. If cells have a specific antigen, they will attract the antibody that fits the antigen. To find out if the antibodies have been attracted to the cells, chemicals will be added that cause the cell to change color only if a certain antibody (and, therefore, the antigen) is present.

Immunohistochemical stains are very useful in identifying certain types of cancers. For example, a routinely processed biopsy of a lymph node may contain cells that clearly appear cancerous, but the pathologist cannot tell whether the cancer started in the lymph node or whether it started elsewhere in the body and spread to the lymph nodes later. If the cancer started in the lymph node, the diagnosis would be lymphoma. If the cancer started in another part of the body and spread to the lymph node, it might be metastatic cancer. This distinction is of great importance to the patient and the doctor, since treatment depends on the type of cancer as well as other factors.

There are hundreds of antibodies used for immunohistochemical tests by laboratories at cancer centers. Some are quite specific, meaning that they react only with one type of cancer. Others may react with a few types of cancer, so several antibodies may be tested to decide what type of cancer it is. By looking at these results along with the cancer's appearance after the biopsy specimen is processed, its location and other information about the patient (age, gender, etc.), it is often possible to classify the cancer in a way that can help the oncologist select the best treatment.

Although immunohistochemical stains are used most often to classify cells, they can also be used to detect or recognize cancer cells. When a large number of cancer cells have spread to a nearby lymph node, these cells are usually recognized easily when the pathologist looks at the lymph tissue under the microscope using routine stains. However, if there are only a few cancer cells in the node, recognizing the cells using only routine stains can be very difficult. This is where immunohistochemical stains can help. Once the pathologist knows the kind of cancer to look for, he or she can choose one or more antibodies known to react with those cells. More chemicals are added so that the cancer cells will change color, which makes them clearly stand out from the normal cells around them. Immunohistochemical stains are generally not used for looking at tissue from lymph node dissections (which remove a large number of nodes), but they are sometimes used in sentinel lymph node biopsies (see the section "Sentinel Lymph Node Mapping and Biopsy").

Another specialized use of imunohistochemical and immunocytochemical stains is to help distinguish lymph nodes that contain lymphoma from those that are swollen from growth of non-cancerous lymphocytes (usually as a response to infection). Certain antigens are present on the surface of immune system cells called lymphocytes. Benign lymph node tissue contains many different types of lymphocytes with a variety of antigens on their surface. In contrast, cancers such as lymphoma start with a single abnormal cell, so that the cancer cells that grow from that cell typically share the chemical features of the first abnormal cell. This is especially useful in the diagnosing lymphoma. If most of the cells in a lymph node biopsy have the same antigens on their surface, this result supports a diagnosis of lymphoma.

Electron Microscopy

The typical medical lab microscope uses a beam of ordinary light to view specimens. A much more complex, larger, and more expensive instrument called an electron microscope uses beams of electrons. The electron microscope's magnifying power is about 1,000 times greater than that of an ordinary light microscope. This degree of magnification is rarely useful in deciding whether a cell is cancerous. But it sometimes helps find very tiny details of a cancer cell's structure that provide clues to the exact type of the cancer. For instance, melanoma, a highly aggressive cancer of the skin, is known to look like other types of cancer when seen under the ordinary light microscope. Although there are some exceptions, melanomas usually can be recognized by certain immunohistochemical stains. In such exceptional cases, the electron microscope may be used to identify tiny bodies located in melanoma cells called melanosomes. This helps to establish the type of cancer, which helps the oncologist choose the best treatment for the patient.

Flow Cytometry

This test is often used to test the cells from bone marrow, lymph nodes, and blood samples. It is very accurate in finding out the exact type of leukemia or lymphoma a person has. It also helps to tell lymphomas from non-cancerous diseases of lymph nodes. A sample of cells from a biopsy, cytology specimen, or blood specimen is treated with special antibodies and passed in front of a laser beam. Each antibody sticks only to certain types of cells that contain the antigens that fit with it. If the sample contains those cells, the laser will cause them to give off light that is then measured and analyzed by a computer.

Analyzing cases of suspected leukemia or lymphoma by flow cytometry uses the same principles explained in the section on immunohistochemistry. Finding the same substances on the surface of most cells in the sample suggests that they came from a single abnormal cell, and are therefore likely to be a cancer. On the other hand, finding several different cell types with a variety of antigens means that the sample is less likely to contain leukemia or lymphoma.

Flow cytometry can also be used to measure the amount of DNA in cancer cells. Instead of using antibodies to detect protein antigens, cells can be treated with special dyes that react with DNA. In this way, one can measure the ploidy of cancer cells, which reflects the amount of DNA they contain. If there's a normal amount of DNA, the cells are said to be diploid. If the amount is abnormal, then the cells are described as aneuploid. Aneuploid cancers of most (but not all) organs tend to be more aggressive than diploid ones.

Another use of flow cytometry is to measure the S-phase fraction, which is the percentage of cells in a sample that are in a certain stage of cell division called the synthesis (or S) phase. The more cells that are in the S-phase, the faster the tissue is growing and the more aggressive the cancer is likely to be.

Image Cytometry

Like flow cytometry, this test uses dyes that react with DNA. But instead of suspending the cells in a stream of liquid and analyzing them with a laser, image cytometry uses a digital camera and a computer to measure the amount of DNA in cells on a microscope slide. Like flow cytometry, image cytometry can determine the ploidy of cancer cells.

Cytogenetics

Normal human cells contain 46 chromosomes (pieces of DNA and protein that control cell growth and function). Some types of cancer have a unique type of abnormal chromosome. Recognizing them helps to identify those types of cancer. This is especially useful in diagnosing some lymphomas, leukemias, and sarcomas. Even if a patient is known to have a certain type of cancer, cytogenetic studies may help predict the outlook for survival. Sometimes the studies can even help predict which chemotherapy drugs the cancer is likely to respond to.

Several types of chromosome changes can be found in cancer cells:

• A translocation means part of one chromosome has broken off and is now located on another chromosome. 
• An inversion means that part of a chromosome is upside down (now in reverse order) but still attached to the right chromosome. 
• A deletion indicates part of a chromosome has been lost. 
• An addition happens when all or part of a chromosome has been duplicated, and too many copies of it are found within the cell.

Cytogenetic testing usually takes about 3 weeks, because the cancer cells must grow in lab dishes for about 2 weeks before their chromosomes are ready to be looked at under the microscope.

Fluorescent in situ hybridization (FISH) is a newer test that is similar to cytogenetic testing. It can find most chromosome changes that can be seen under a microscope in standard cytogenetic tests. It can also find some changes too small to be seen with usual cytogenetic testing. FISH uses special fluorescent dyes that only attach to specific parts of certain chromosomes. FISH can find chromosome changes such as translocations, which are important to help classify some kinds of leukemia. This test can also recognize when there are too many copies of a certain gene (gene amplification), which can help choose the best treatment for some women with breast cancer. Unlike standard cytogenetic tests, it is not necessary to grow cells in laboratory dishes before doing FISH. That means FISH results are available much sooner, usually within a few days.

Molecular Genetic Studies

DNA and RNA tests can be used to find most of the translocations that are found by cytogenetic tests. They can also find some translocations involving parts of chromosomes too small to be seen with usual cytogenetic testing under a microscope. This type of advanced testing can help classify some leukemias and, less often, some sarcomas and carcinomas. These tests are also useful after treatment to find small numbers of remaining leukemia cancer cells that may be missed under a microscope.

Molecular genetic tests can also identify mutations (abnormal changes) in certain areas of DNA that are responsible for regulating cell growth. Some of these mutations may cause cancers to be especially aggressive in growing and spreading. In some situations, identifying certain mutations can help doctors choose treatments that are more likely to work.

Certain substances called antigen receptors appear on the surface of immune system cells called lymphocytes. Normal lymph node tissue contains lymphocytes with many different antigen receptors, which help the body respond to infection. Some types of lymphoma and leukemia, however, start from a single abnormal lymphocyte, so all their cells have the same antigen receptor. Lab tests of the DNA on each cell's antigen receptors are a very sensitive way to diagnose and classify these cancers.

Some gene mutations can be inherited from parents and cause a person to have a greater risk of developing certain cancers. Unlike acquired gene mutations that only affect the abnormal cells of the tumor, inherited mutations affect all cells of a person's body. These inherited mutations can often be identified by genetic testing on blood samples. Genetic counseling and testing may be recommended for some people with a strong family history of cancer. Because these tests do not analyze the cancerous tissue, they are not discussed further in this document. For more information, see the American Cancer Society document, Genetic Testing: What You Need to Know.

Polymerase chain reaction (PCR) is a very sensitive molecular genetic test for finding specific DNA sequences, such as those occurring in some cancers. Reverse transcriptase PCR (RTPCR) is a method for detecting small amounts of RNA, a substance related to DNA that is needed for cells to produce proteins. There are specific RNAs for each protein in our body. RTPCR can be used to find and classify cancer cells.

RTPCR tests to detect cancer cells look for the RNA sequences that are responsible for making substances found in cancer cells but not in most normal cells. An advantage of this test is that it can detect very small numbers of cancer cells in the blood or tissue samples that would be missed by other tests. RTPCR is already used routinely for detecting certain kinds of leukemia cells that remain after treatment, but its value for more common types of cancer is less certain. The disadvantage is that doctors are not always sure whether having a few cancer cells in the bloodstream or a lymph node means that a patient will actually develop distant metastases that grow enough to cause symptoms or affect survival. In treating patients with most common cancer types, it is still uncertain whether recognizing a few cancer cells with this test should be a factor in choosing treatment options.

RTPCR can also be used to sub-classify cancer cells. Some RTPCR tests measure levels of one or even several RNAs at the same time. By comparing the levels of important RNAs, doctors can sometimes predict whether a cancer is likely to be more or less aggressive than would be expected based on its microscopic appearance alone. Sometimes these tests can help predict whether a cancer will respond to certain treatments.

Gene expression microarrays are miniaturized devices that are similar in some ways to computer chips. The advantage of this technology is that relative levels of hundreds or even thousands of different RNA molecules from one sample can be compared at the same time. Recent studies have found that this information can sometimes help predict a patient's prognosis or response to certain treatments. Although this is a very active area of research, doctors are still conducting studies to learn how this information should guide their treatment recommendations. For now, most cancer treatment guidelines do not recommend routine use of these tests.

How long does biopsy and cytology testing take?

The uncertainty you feel waiting for biopsy and cytology test results can be a source of much anxiety. Not knowing when the results will be ready and not understanding why testing sometimes takes longer than expected can cause extra concern.

Routine biopsy and cytology results may be ready as soon as 1or 2 days after the sample is received in the laboratory. But there are many reasons why some cases take considerably longer to complete.

Often, there are "technical" reasons for delays in reporting. For example, bone and other hard tissues that contain lots of calcium need to be specially handled. These tissues must be treated with strong acids or other chemicals to remove the minerals so that the tissue becomes soft enough to be thinly sectioned (sliced) on the microtome. Another technical reason for delay is that the formalin solution used for preserving tissues takes longer to penetrate samples with lots of fatty tissue (such as breast biopsies). An extra day of fixation (formalin treatment) is sometimes necessary. Large samples, such as those resulting from removal of an entire organ, might also require more than one day for the formalin to penetrate the tissue. If formalin does not completely penetrate the sample, cells may appear disturbed under the microscope and testing is more difficult and/or less accurate.

For most large samples, only selected areas are processed and examined under the microscope. After the first sections of tissue are seen under the microscope, the pathologist may want to look at more sections for an accurate diagnosis. In these cases, processing of extra pieces of tissue may be needed. Or the lab may need to make more slices of the tissue that has already been embedded in wax blocks. Either of these situations can add 1or 2 days to the testing time.

Although most cancers can be found by looking at routinely stained sections, other studies such as those already described may be needed for some specimens. For example, histochemical stains or immunoperoxidase stains usually delay a case for another day. Other advanced studies like flow cytometry, electron microscopy, and molecular pathology techniques can take even longer, sometimes days, before results are ready.

Another important reason for delaying a pathology report is that the pathologist may seek a second opinion from an expert. Unlike some chemical tests done in the laboratory that measure the amount of a specific substance or look at whether a substance is present or absent, testing tissue or cell samples for cancer is based on the professional opinion of the pathologist who looks at the sample under the microscope. Although the abnormal features of some cancers are obvious, some cases have features that are very difficult to recognize. Also, pathologists are often understandably reluctant to diagnose certain very rare types of cancer without a second opinion from an expert who specializes in that area. There are pathology experts specializing in almost every organ system (digestive, head and neck, breast, bone, reproductive, etc.). When difficult or rare cases are encountered, slides are usually sent to experts by overnight mail. Such review can delay the case for several more days.

Finally, patients should realize that delays might occur for reasons that are neither technical nor medical. For example, entering the report into the computer takes time. Some labs send results directly to doctors’ office computer systems or fax machines, but a hospital mail system or US mail is still often used and can delay the results.

What can you do to learn more about your pathology results?

Pathology results have a key role in making decisions about treatment, and many patients want to learn more about their test results. You should feel free to ask your doctors to explain these results in a way that you can understand. You will want to focus on how the results influence treatment options and help predict your outlook for survival. Some pathologists will speak with you to help you understand your pathology reports. But some other pathologists believe that your oncologist, primary care doctor, or other doctors are better able to explain the results because they know more of your overall medical situation. Also, doctors who already know you well are often best able to discuss the complex personal issues affected by your pathology results.

You may request copies of your pathology reports, and you may find it useful to keep a folder or notebook with your pathology, radiology, and other test results. If you see more doctors in the same hospital where your cancer was diagnosed, the new doctors will have access to the original pathology report and other medical records. If consulting doctors (such as those sought for second opinions) practice at other facilities, it is usually necessary to send copies of pathology reports and other medical records. Usually you can simply sign a release form to have the copies sent, but it is helpful if you keep an original copy to share with the new doctor in case a report is not available. You will always want to get back the original for those times you may need it again.

Some cancer centers have a policy requiring that microscope slides of the patient's cancer be reviewed by the pathologists at their own institution. Some pathology laboratories will give copies of microscope slides to you if you are about to visit another cancer center for a second opinion or consultation. Other laboratories prefer to mail the slides directly to the consulting cancer center's pathology department.

If you or your doctors have any concerns about your pathology diagnosis, you can have your microscope slides reviewed by a consulting pathologist for a second opinion. Your oncologist or surgeon or the pathologist who first looked at your biopsy or cytology sample can often suggest a consultant with special qualifications in examining samples such as yours. Or you can have your slides sent to the pathology department of a medical school or cancer center you have confidence in.

What information is included in a pathology report?

The pathology report of surgical specimens is often quite long and complex. It is typically divided into a number of subheadings.

Patient, doctor, and specimen identification: The general identifying information includes the patient's name, medical record number issued by the hospital, the date when the biopsy or surgery was performed and the unique number of the specimen issued in the laboratory.

Clinical information: The next portion of the report often contains information about the patient provided by the doctor who removed the tissue sample. Such information may include a pertinent medical history and special requests made to the pathologist. For example, if a lymph node sample is being removed from a patient already known to have cancer in another organ, the doctor will note the type of the original cancer. This information is often useful in guiding the pathologist's selection of special studies that may be needed to find out whether any cancer in that lymph node is a metastasis from the prior cancer or is a new cancer that started in the lymph node.

Gross description: The next part of the report is called the gross description. The medical meaning of "gross" differs from the common use of the word, and refers to features that can be identified without a microscope (by simply looking at, measuring, or feeling the tissue).

For a small biopsy, this description is a few sentences listing its size color, and consistency. This section also records the number of tissue-containing cassettes submitted for processing.

Larger biopsy or tissue specimens, for example, a mastectomy for breast cancer, will have much longer descriptions including the size of the entire breast, size of the cancer, how close the cancer is to the nearest surgical margin or edge of the specimen, how many lymph nodes were found in the underarm area, and the appearance of non-cancerous breast tissue. A summary of exactly where tissue was taken from for processing is included.

For cytology specimens, the gross description is very short and usually notes the number of slides or smears made by the doctor. If the sample is a body fluid, its color and volume are noted.

Microscopic description: This description records what the pathologist saw under the microscope. The appearance of the cancer cells, how they are arranged together, and the extent to which the cancer penetrates nearby tissues in the specimen are usually included in the microscopic description. For typical cases of common cancers or for benign tissues, a microscopic description may not be included in the report. Results of any additional studies (histochemical stains, flow cytometry, etc.) performed in the case are noted in the microscopic description or in a separate section.

Diagnosis: The most important part of the pathology report is the final diagnosis. It is, in essence, the "bottom line" of the testing process, although this section may appear at the bottom or the top of the page. The patient's doctor relies upon this final diagnosis to help in choosing the best treatment options for the patient. If the diagnosis is cancer, this section will note the exact type of cancer that is present and will usually include the cancer's grade.

Comment: After the final diagnosis is made, the pathologist may wish to add more information for the doctors taking care of the patient. The comment section is often used to clarify a concern or recommend further testing.

Summary: Some pathology reports for cancers contain a summary of findings most relevant to making treatment decisions.

Additional Resources

More Information From Your American Cancer Society

We have selected some related information that may also be helpful to you. These materials may be ordered from our toll-free number or found on our Web site, at www.cancer.org.

After Diagnosis: A Guide for Patients and Families (also available in Spanish)

Choosing a Doctor and a Hospital (also available in Spanish)

Talking with Your Doctor (also available in Spanish)

Surgery (also available in Spanish)

Understanding Chemotherapy (also available in Spanish)

Understanding Radiation Therapy (also available in Spanish)

National Organizations and Web Sites*

In addition to the American Cancer Society, other sources of patient information and support include:

National Cancer Institute
Telephone: 1-800-4-CANCER (1-800-422-6237), TTY: 1-800-332-8615
Internet Address: http://www.cancer.gov

College of American Pathologists
Internet Address: http://www.MyBiopsy.org

*Inclusion on this list does not imply endorsement by the American Cancer Society.

No matter who you are, we can help. Contact us anytime, day or night, for information and support. Call us at 1-800-ACS-2345 or visit www.cancer.org.

Revised: 12/10/2007