With so many types of lymphoma, there’s a wide range of corresponding lab tests to detect their presence in patients. Here, CLP takes a look at testing for the most common type of lymphoma (diffuse large B-cell lymphoma) and leukemia (chronic lymphocytic leukemia) in adults.

Eric Hsi, section head of hematopathology, Department of Clinical Pathology, Cleveland Clinic, says labs will often detect an abnormally high lymphocyte count from a patients’ complete blood count (CBC) and leukocyte differential. A lab discovers the presence of elevated lymphocytes (white blood cells that accumulate when there is chronic inflammation) when the hematology analyzer flags the samples as abnormal. A blood smear is then made and sent to a laboratory technologist and then to a pathologist for review under the microscope. The high number of lymphocytes could be an indicator of chronic lymphocytic leukemia (CLL), a malignancy of the lymphocytes (a type of white blood cell). CLL is characterized by a slow, progressive increase of these cells in the blood and the bone marrow. These cancerous cells replace bone marrow and can invade other tissues such as lymph nodes and the spleen. They may cause immunosuppression and failure of the bone marrow due to infiltration. The bone-marrow failure manifests as anemia (deficiency of red blood cells that carry oxygen to tissues) and thrombocytopenia (deficiency of platelets, resulting in high risk of bleeding). Hsi adds that some CLL patients are asymptomatic or have only mild nonspecific symptoms, such as fatigue.

In cases in which an elevated lymphocyte count is present in an adult patient, a flow-cytometry test may be indicated to characterize the lymphocytes, Hsi says. “That would be the next step. Upon reviewing a blood smear visually under the microscope and seeing increased numbers of mature-appearing lymphocytes (characteristic of CLL), a pathologist might consult with the patient’s primary physician and recommend flow cytometry. Flow cytometry involves testing cells for many diagnostically useful antigens using fluorescently labeled antibodies. Antibodies bind to cells if the antigen to which the antibody is directed is present on the cells. The cells will then fluoresce when interrogated by a laser beam that excites the fluorescent label attached to the antibody,” Hsi says. “The exact pattern of positivity or negativity for these diagnostically useful antigens is characteristic for different types of leukemias. If the clinical laboratory has its own specialized flow-cytometry laboratory, the pathologists might be able to use the leftover blood from the CBC sample, streamlining the diagnostic process. Alternatively, the patient would be called to give another blood sample specifically for flow cytometry. After reviewing the blood smear microscopically and interpreting the flow-cytometry data, the pathologist can diagnose CLL.”

Hsi says that diagnosing lymphoma is a little different in that most of those patients are being seen because of symptoms such as enlarged lymph nodes that may be painless or associated with fatigue, night sweats, or fever. “If an enlarged lymph node is detected by a physician and it does not appear related to a reactive cause such as infection, a biopsy might be ordered. Most enlarged lymph nodes are benign, not malignant, and so are treated with antibiotics,” Hsi says. However, if the enlargement does not go away, a lymph-node biopsy is indicated. Tissue from the suspect area is biopsied and processed in the pathology lab. It is fixed, often in formaldehyde, and embedded in paraffin wax. Then, histologic slides are made for review by a pathologist. In many laboratories, the pathologist also takes a small piece of the lymph node prior to fixation and sends it for flow cytometry and cytogenetic analysis to help determine whether it is lymphomatous in much the same way as for CLL. A portion may also be saved in a fresh-frozen state for subsequent molecular genetic studies that may assist in diagnosis and classification of lymphoma, Hsi says. Some pathologists do not send this fresh tissue and fresh-frozen tissue. Hsi says this is an unfortunate occurrence, since these studies (flow cytometry, cytogenetics, and molecular genetic tests) are often needed for an accurate diagnosis. This may occur for a number of reasons, such as placing the tissue in fixative without notifying a pathologist to examine the fresh tissue at the time of the biopsy.

Lymphoma Defined
There are two main types of lymphomas. Hodgkin lymphoma (also known as Hodgkin’s lymphoma, Hodgkin disease, or Hodgkin’s disease) is named after Thomas Hodgkin, MD, who first described it as a new disease in 1832. All other types of lymphoma are called non-Hodgkin lymphomas.

These two types of lymphoma can usually be distinguished from each other by examining the cancerous tissue under a microscope. In some cases, more tests to identify specific chemical components of the lymphoma cells may be needed, according to the American Cancer Society.

The more common Hodgkin disease is a cancer that starts in lymphatic tissue. Lymphatic tissue includes the lymph nodes and related organs that are part of the body’s immune and blood-forming systems, such as the spleen and bone marrow. Lymph nodes are found underneath the skin in the neck, under the arms, and in the groin, as well as in many other places in the body, such as inside the chest, abdomen, and pelvis.

Lymph nodes make and store lymphocytes, which are a type of infection-fighting white blood cell. The two types of lymphocytes are known as B lymphocytes (B cells) and T lymphocytes (T cells).

The Lymphoma Information Network says that non-Hodgkin’s lymphomas caused by malignant (cancerous) B-Cell lymphocytes represent a large subset (about 85% in the United States) of the known types of lymphoma. The other two subsets are T-cell lymphomas and lymphomas, where the cell type is the natural killer cell or unknown). B cells undergo many changes in their life cycle dependent on complex signaling processes between cells and interaction with foreign substances in the body. Various types of lymphoma or leukemia can occur in the B-cell life cycle.

New Markers Better Identify Patients at Risk
With newer, more sophisticated markers, physicians and labs have a better idea about the characteristics of lymphomas and leukemias, and can better determine which patients with a particular leukemia or lymphoma are at risk for a more aggressive disease course, Hsi says. This may be done by the use of chromosomal analysis, molecular genetic studies, or specific protein-expression tests. One test, the ZAP-70 expression test in CLL, came from gene-expression profiling data, he says. ZAP-70 is normally expressed in T cells and natural killer cells and is important in T-cell receptor signaling. In some cases of CLL, it appears to be abnormally expressed in the malignant B-cells and identifies patients with a more aggressive course. With regard to chromosomal abnormalities, loss of a portion of chromosome 17 has also been shown to identify a group of CLL patients with a very aggressive form of CLL. Also, he adds, because of targeted therapies such as anti-CD20 or anti-CD52 monoclonal antibodies, pathologists can look for these particular targets on the malignant cells to provide evidence that the antibodies will be effective.

“With diffuse large B-cell lymphoma, genetic-profiling studies have shown that the tumors can be segregated into ones that resemble normal germinal center and nongerminal center B-cells. Other types are also being discovered. These divisions appear to have prognostic significance, meaning that diffuse large B-cell lymphoma can be divided into more than one subtype and that their clinical courses are different. The challenge is to see if such models are still accurate and meaningful as new therapies are developed,” Hsi says.

According to Daniel Arber, MD, professor of pathology and associate chair for hemopathology, Stanford University School of Medicine, there are two main ways to diagnose diffuse large B-cell lymphoma when performing tissue biopsies. The less invasive method is performing a fine needle aspiration, whereby a needle is inserted in a large lymph node and fluid is aspirated. If suspect for lymphoma, flow cytometry is used to determine if clonal B cells are present. This procedure is done in a hematology lab, using a flow cytometer to run the cells through. They’re hit with a laser, which lights up the antibodies and tells the pathologist what antigens are present, he says. “We’re looking for B cells, because about 85% of lymphomas are B cells; the normal cells are T cells,” Arber says. When clonal B cells are found, further correlation is done by making slides for a cytology review by a pathologist. “We want to see if the cells are too large. The size of the cell is the tip-off,” he says.

If such enlarged cells are discovered, a report is issued and the patient is referred to an oncologist for treatment with chemotherapy. B-cell lymphoma is often treated with CHOP-R (the acronym for a particular chemotherapy regime used in lymphoma treatment, consisting of cyclophosphamide, hydroxydoxorubicin, Oncovin®, and prednisone. Combined with the monoclonal antibody rituximab, it is known as CHOP-R). The latter, Arber says, has the antibody for B-cell antigens, and thus targets the B-lineage tumor cells.

Arber notes that lymphoma arises from lymphocytes that take off, or grow, out of control. “They’re like rogue cells,” he says. The basic treatment, chemotherapy, has about a 40% to 50% cure rate for diffuse large B-cell lymphoma.

Testing for leukemia is similar, but a different sample is often used, Arber says. The test involves doing a bone marrow aspiration to test for B-cell or T-cell types. A similar test involves drawing blood and analyzing it using flow cytometry at specialty labs.

New Testing Methods
Richard Furman, MD, assistant professor, Center for Lymphoma and Myeloma, Weill Medical College of Cornell University, says a good deal of attention has been paid to DNA microarray analysis, a research tool for examining genes transcribed in a cell. The DNA microarray can help differentiate diffuse large B-cell lymphomas into one of two groups: those of germinal center B-cell (GBC) phenotype or that of an activated B-cell (ABC) phenotype. This difference is associated with a difference in terms of prognosis, but at the current time, there are no data to suggest an advantage to treating these patients differently. While DNA microarray is not commercially available at this time, there are surrogate markers detectable by immunohistochemistry (IHC), including: CD10, MUM 1, Bcl-2 and Bcl-6, he says. Based upon the expression of these proteins, diffuse large B-cell lymphomas can be assigned to either group.

When a patient presents for an evaluation of possible lymphoma, the tests performed on the tissue specimen will be determined by whether the specimen is blood, bone marrow, lymph node, or other tissue. While blood and bone marrow lend themselves to analysis by flow cytometry because the cells are already in a suspension, lymph nodes can be processed to enable them to be analyzed by flow cytometry. The tissue is, in essence, ground up and the cells are then suspended. IHC, on the other hand, can be done on tissue slides. The tissue to be examined just needs to be sliced thin enough to be viewed under the microscope. The World Health Organization’s (WHO) Classification of Lymphoid Neoplasms used immunologic, morphologic, and genetic features of the tumor to classify which type of lymphoma it is. The importance of this classification is to enable better prediction of clinical behavior and outcome, and to guide therapy.

Flow cytometry and IHC serve as the means for determining the phenotype of the lymphoma cells, which is the characterization of the proteins on its surface. IHC uses antibodies with enzymes that catalyze reactions; the resulting color changes to determine whether the protein is present on the cell. Flow cytometry uses antibodies tagged with a fluorochrome that is then detected with a laser. The genetic features of the lymphoma are analyzed by polymerase chain reaction (PCR) and interphase fluorescent in situ hybridization (iFISH). PCR enables the detection of clonal rearrangements of the immunoglobulin and T-cell receptor genes, as well as fusions between genes that play roles in lymphomagenesis. These genetic fusions can also be detected by iFISH, which uses fluorescent probes for genes. Each gene is highlighted by a different color. When the genes are fused, the signals from the probes overlap sufficiently to give a different color. Likewise, when a gene is broken into two by a translocation, one will be able to see two colors instead of the one combination, with a reason to be evaluated for diagnosis, Furman says. A biopsy or a bone-marrow markup may be performed. Using the WHO’s classification system, a thin slice of tissue is taken and its cells are examined under a microscope. The molecules on the surface of the cell are analyzed using IHC and flow cytometry, Furman says.

Cytogenetics is not an important factor in lymphoma testing, but finding for two genes inappropriately brought together can be an indicator. It all starts, though, with a histological review, Furman says. For leukemia testing, flow cytometry is widely used, because blood lends itself nicely to such a test, he says.

Furman notes that there’s a large variance in labs and that the human component is important. “A lot depends on the quality of the pathologist,” Furman says.

What About the Future?
Arber says there is a lot of discussion about the future of gene-array testing. Studies in the past 5 years have shown that gene arrays can divide diffuse large B-cell lymphoma into a couple of biologic subtypes that have clinical significance (one type has longer survival with conventional therapy compared to the other). “It looks like we are still a long way from offering those arrays as a diagnostic test in clinical laboratories, but we have identified immunohistochemical markers that can subcategorize them in a similar fashion to the much more complicated gene arrays,” Arber says. IHC, which is already used for the tissue biopsy diagnosis of diffuse large B-cell lymphoma, is being expanded to also aid in the determination of prognosis (just by adding a few more antibody studies), Arber notes.

Gary Tufel is a contributing writer for Clinical Lab Products.