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Molecular tests for the diagnosis and classification of canine lymphoma
VOLUME 18 ISSUE 8 AUGUST 2005
Advances in Small Animal Medicine and Surgery Molecular Tests for the Diagnosis and Classification of Canine Lymphoma Linda M. Berent, DVM, PhD Diplomate, ACVP (Clinical Pathology) Clinical Assistant Professor University of Missouri Columbia, MO Lymphoma is one of the most common malignancies in our canine patients, but the diagnosis of this disease is not always straightforward. A clinical pathologist is often called upon to examine populations of cells in blood, fluids, or tissues and make a diagnostic decision. In most cases, this diagnosis can easily be made based on the presence of a monomorphic population of large immature lymphocytes. One of the most frustrating scenarios a pathologist and practitioner must face is when the patient has non-specific clinical signs and a cytology report contains the words “suggestive of, but not diagnostic for, lymphoma.” This often happens in hemogram reports when there is a chronic or mild lymphocytosis, but the cells are well differentiated and do not “look” malignant. Or, it may happen when a fluid analysis does not fit clinically with a chylous effusion, but in which there are just too many lymphocytes. Another situation may be a tissue aspirate in which one sees too many lymphocytes, but it is a mixed population. In each of these cases, the patient may very well have an exaggerated immune response which has stimulated the lymphocytes; however, they may be in the early stages of lymphoma or leukemia. A timely and accurate diagnosis of the disease in these patients will result in decreased suffering on the part of the patient and decreased anxiety on the part of the owner. Recent advances in the molecular diagnosis of lymphoma have now moved out of the research lab and are being marketed to veterinarians. Both the PCR for
antigen receptor rearrangement (PARR) and flow cytometry are commercially available at the Colorado State University Veterinary Diagnostic Laboratory and can be run on a variety of fluids.1 These tests have great potential to aid in the diagnosis and characterization of canine lymphoma.
“Both techniques will currently allow a veterinarian to give a more accurate prognosis and survival estimate.” FLOW CYTOMETRY Commonly, a pathologist will use immunohistochemical stains to provide an immunophenotype and determine if a lymphoid population is a B-cell or a Tcell population. Flow cytometry is similar to traditional immunophenotyping in that the proteins present on the outer layer of the cells are analyzed. This technique gives more information on the lineage of the cells as it uses a larger panel of antibodies to characterize the proteins expressed within a population. Flow cytometry is also more helpful in characterizing leukemias of myeloid origin. As more information is gathered on the various immunophenotypic and morphologic subtypes within canine lymphoma and leukemia, veterinarians may be able to customize treatment, as is done with human patients. Both techniques will currently allow a veterinarian to give a more
accurate prognosis and survival estimate, as it is well established that dogs with B-cell lymphoma have a more favorable outcome.2 PARR TESTING The PARR test is used on a tissue sample to see if a clonal population of lymphocytes is present. This test looks specifically at the DNA in the sample for rearrangements in either the T-cell receptor gene or the B-cell immunoglobulin gene to determine if all the cells have these receptors arranged in the same manner. In a reactive population, one would expect a variety of different gene rearrangements which would lead to a polyclonal population.
Advances
Page 2
In a neoplastic lymphoid population, all of the cells are theoretically derived from one malignant precursor and will all have the same gene rearrangement.3 A recent review article by Drs. Paul and Anne Avery outlines the usefulness and limitations of molecular diagnostic techniques.4 The tests are validated for use in the dog; feline tests are still in the experimental phase. PARR may also be used in the staging and monitoring of lymphoma patients. One study using dogs previously diagnosed with lymphoma showed that PARR was 2.5 times more likely to identify malignant lymphocytes in a peripheral blood sample than by simply scanning a blood smear using traditional microscopy.5 The PARR technique is best used as an additional diagnostic tool rather than a replacement for traditional methods and for the following practical considerations: a) The best use of the PARR test is in determining if a lymphocyte population is reactive or neoplastic, but false-positive results are still possible, particularly in Ehrlichia canis infected patients. Caution should be used in the interpretation of the PARR test if you suspect the animal has E. canis. b) This test should be able to distinguish a B-cell from a T-cell lymphoma in most animals, but in rare cases may
give erroneous results. The phenotype of the cells is best acquired from immunohistologic staining or flow cytometry. These techniques tell you what proteins the malignant cells are producing, not just what genes are rearranged. Some myeloid leukemias and B-cell lymphomas have been known to rearrange their T-cell receptor gene and would give a false positive on a PCR for T-cell clonality. c) Sample cellularity must be adequate. This test is so sensitive that it can give a false positive if too few lymphocytes are present. After all, if only one lymphocyte is present, it will be a clonal population. REFERENCES 1. http://www.dlab.colostate.edu/images/ clinicalimmunoform0704.pdf. Colorado State University Veterinary Diagnostic Laboratory – Clinical Immunology section. 2. Ponce F, Magnol JP, Ledieu D, et al. Prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy. Vet J 2004;167:158166. 3. Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003;40:32-41. 4. Avery PR, Avery AC. Molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states. Vet Clin
Pathol 2004;33:196-207. 5. Keller RL, Avery AC, Burnett RC, et al. Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement. Vet Clin Pathol 2004;33:145-149.
Advances in Small Animal Medicine and Surgery Molecular Tests for the Diagnosis and Classification of Canine Lymphoma Linda M. Berent, DVM, PhD Diplomate, ACVP (Clinical Pathology) Clinical Assistant Professor University of Missouri Columbia, MO Lymphoma is one of the most common malignancies in our canine patients, but the diagnosis of this disease is not always straightforward. A clinical pathologist is often called upon to examine populations of cells in blood, fluids, or tissues and make a diagnostic decision. In most cases, this diagnosis can easily be made based on the presence of a monomorphic population of large immature lymphocytes. One of the most frustrating scenarios a pathologist and practitioner must face is when the patient has non-specific clinical signs and a cytology report contains the words “suggestive of, but not diagnostic for, lymphoma.” This often happens in hemogram reports when there is a chronic or mild lymphocytosis, but the cells are well differentiated and do not “look” malignant. Or, it may happen when a fluid analysis does not fit clinically with a chylous effusion, but in which there are just too many lymphocytes. Another situation may be a tissue aspirate in which one sees too many lymphocytes, but it is a mixed population. In each of these cases, the patient may very well have an exaggerated immune response which has stimulated the lymphocytes; however, they may be in the early stages of lymphoma or leukemia. A timely and accurate diagnosis of the disease in these patients will result in decreased suffering on the part of the patient and decreased anxiety on the part of the owner. Recent advances in the molecular diagnosis of lymphoma have now moved out of the research lab and are being marketed to veterinarians. Both the PCR for
antigen receptor rearrangement (PARR) and flow cytometry are commercially available at the Colorado State University Veterinary Diagnostic Laboratory and can be run on a variety of fluids.1 These tests have great potential to aid in the diagnosis and characterization of canine lymphoma.
“Both techniques will currently allow a veterinarian to give a more accurate prognosis and survival estimate.” FLOW CYTOMETRY Commonly, a pathologist will use immunohistochemical stains to provide an immunophenotype and determine if a lymphoid population is a B-cell or a Tcell population. Flow cytometry is similar to traditional immunophenotyping in that the proteins present on the outer layer of the cells are analyzed. This technique gives more information on the lineage of the cells as it uses a larger panel of antibodies to characterize the proteins expressed within a population. Flow cytometry is also more helpful in characterizing leukemias of myeloid origin. As more information is gathered on the various immunophenotypic and morphologic subtypes within canine lymphoma and leukemia, veterinarians may be able to customize treatment, as is done with human patients. Both techniques will currently allow a veterinarian to give a more
accurate prognosis and survival estimate, as it is well established that dogs with B-cell lymphoma have a more favorable outcome.2 PARR TESTING The PARR test is used on a tissue sample to see if a clonal population of lymphocytes is present. This test looks specifically at the DNA in the sample for rearrangements in either the T-cell receptor gene or the B-cell immunoglobulin gene to determine if all the cells have these receptors arranged in the same manner. In a reactive population, one would expect a variety of different gene rearrangements which would lead to a polyclonal population.
Advances
Page 2
In a neoplastic lymphoid population, all of the cells are theoretically derived from one malignant precursor and will all have the same gene rearrangement.3 A recent review article by Drs. Paul and Anne Avery outlines the usefulness and limitations of molecular diagnostic techniques.4 The tests are validated for use in the dog; feline tests are still in the experimental phase. PARR may also be used in the staging and monitoring of lymphoma patients. One study using dogs previously diagnosed with lymphoma showed that PARR was 2.5 times more likely to identify malignant lymphocytes in a peripheral blood sample than by simply scanning a blood smear using traditional microscopy.5 The PARR technique is best used as an additional diagnostic tool rather than a replacement for traditional methods and for the following practical considerations: a) The best use of the PARR test is in determining if a lymphocyte population is reactive or neoplastic, but false-positive results are still possible, particularly in Ehrlichia canis infected patients. Caution should be used in the interpretation of the PARR test if you suspect the animal has E. canis. b) This test should be able to distinguish a B-cell from a T-cell lymphoma in most animals, but in rare cases may
give erroneous results. The phenotype of the cells is best acquired from immunohistologic staining or flow cytometry. These techniques tell you what proteins the malignant cells are producing, not just what genes are rearranged. Some myeloid leukemias and B-cell lymphomas have been known to rearrange their T-cell receptor gene and would give a false positive on a PCR for T-cell clonality. c) Sample cellularity must be adequate. This test is so sensitive that it can give a false positive if too few lymphocytes are present. After all, if only one lymphocyte is present, it will be a clonal population. REFERENCES 1. http://www.dlab.colostate.edu/images/ clinicalimmunoform0704.pdf. Colorado State University Veterinary Diagnostic Laboratory – Clinical Immunology section. 2. Ponce F, Magnol JP, Ledieu D, et al. Prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy. Vet J 2004;167:158166. 3. Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003;40:32-41. 4. Avery PR, Avery AC. Molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states. Vet Clin
Pathol 2004;33:196-207. 5. Keller RL, Avery AC, Burnett RC, et al. Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement. Vet Clin Pathol 2004;33:145-149.