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Genetic Relatedness of Lymphoid Malignancies: Transformation of Chronic Lymphocytic Leukemia as a Model

Kenneth A. Foon, MD; Raghu Thiruvengadam, MD; Alan Saven, MD; Zale P. Bernstein, MD; and Robert Peter Gale, MD, PhD
[+] Article and Author Information

From the Markey Cancer Center and the Division of Hematology and Oncology, Department of Medicine, University of Kentucky, Lexington, Kentucky; Scripps Clinic and Research Foundation, La Jolla, California; Roswell Park Cancer Institute, Buffalo, New York; University of California at Los Angeles, Los Angeles, California. Requests for Reprints: Kenneth A. Foon, MD, Markey Cancer Center, 800 Rose Street, Room 140, Lexington, KY 40536-0093. Acknowledgments: The authors thank Drs. John Spinosa and Douglas Ellison for histopathologic materials, Dr. Richard McPherson for the Southern blot, and Dr. Ann Marie Block for the karyotype.


Copyright 2004 by the American College of Physicians


Ann Intern Med. 1993;119(1):63-73. doi:10.7326/0003-4819-119-1-199307010-00011
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Objective: Studies concerning the genetic relatedness between chronic lymphocytic leukemia and the more aggressive B-cell cancers that develop in about 10% of affected persons were reviewed. These B-cell cancers include large B-cell lymphoma (the Richter syndrome), prolymphocytic transformation, acute lymphoblastic leukemia, and multiple myeloma. Two possible relations were evaluated: development from the chronic lymphocytic leukemia clone (clonal evolution) and development of a genetically unrelated, independent second cancer.

Data Analysis: Analysis of genetic relatedness between the two cancers considered concordance for immunoglobulin gene rearrangements, for immunoglobulin isotypes and idiotypes, and for cytogenetic abnormalities.

Conclusions: In the case of large B-cell lymphoma, generally thought to arise from the chronic lymphocytic leukemia clone, approximately one half of the patients had genetically unrelated cancers. In prolymphocytic transformation, all cases studied appeared to evolve from the chronic lymphocytic leukemia clone. The few studies of acute lymphoblastic leukemia and multiple myeloma showed genetic relatedness in some cases and unrelatedness in others. These data indicate that progression to more aggressive B-cell cancers in persons with chronic lymphocytic leukemia can result from either clonal evolution or from an independent transforming event.

Figures

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Figure 1.
Scheme of development of chronic lymphocytic leukemia.leftright

Transformation of one B cell to produce chronic lymphocytic leukemia is shown. Clonality is confirmed by analysis of immunoglobulin genes (Southern blotting), antibodies (anti-isotypic and anti-idiotypic), or chromosome analysis. The karyotype in the Figure shows a trisomy 12, and the Southern blot shows the same germline in the normal ( ) and the patient's ( ) peripheral blood cells (bar). A single immunoglobulin gene rearrangement is identified in the patient's cells () but not in the normal cells. Anti-idiotype antibodies are depicted as red with a green fluorescein tag and show specific reactivity with the immunoglobulin on the chronic lymphocytic leukemia cells. Determining genetic relatedness of the chronic lymphocytic leukemia clone to large B-cell lymphoma, prolymphocytic transformation, acute lymphoblastic leukemia, or multiple myeloma requires reactivity with the same anti-idiotype antibody, showing identical immunoglobulin gene rearrangements or identical karyotype.

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Figure 2.
Southern blotting.arrows

High-molecular-weight DNA is isolated from fresh, frozen, or fixed tissues; digested with restriction endonuclease enzymes; and electrophoresed overnight in agarose gel to separate DNA fragments of different sizes. These fragments are then transferred to nitrocellulose filters by Southern blotting and the filters are hybridized with Phosphorus-32-labeled DNA probes homologous to immunoglobulin heavy- and light-chain constant region genes. Autoradiography is then done. Labeled bands are detected on roentgenographic films only when clonal immunoglobulin gene rearrangement is present. The Southern blot shown is an EcoRI restriction enzyme. The normal (N) column shows a germline (bar), and the patient's (P) column also shows a gene rearrangement ( ) in addition to the germline.

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Figure 3.
Generation of anti-idiotype antibodies.

Immunoglobulin is isolated from a human B-cell tumor by fusing it with immunoglobulin nonsecreting murine myeloma cells using polyethylene glycol (PEG). The immunoglobulin is purified, mixed with complete Freund adjuvant (CFA), and used to immunize BALB/c mice. Immunized mice are killed, and B cells from the spleen are fused with immunoglobulin nonsecreting murine myeloma cells. Supernatants from the hybridomas are tested for anti-idiotype antibodies by screening for reactivity against immunoglobulin from the original human B-cell tumor. Supernatants with antibodies reacting only with the immunoglobulin from the original B-cell tumor are identified, subcloned, and retested for specific anti-idiotype reactivity. After anti-idiotype antibodies are identified, they can be used to identify B cells by flow cytometric or immunochemical techniques. With rare exceptions, they react specifically with the original B-cell tumor cells and not with normal B cells or other B-cell tumors.

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Figure 4.
Cytogenetic analysis.

Cells from blood, bone marrow, or lymph nodes are stimulated to divide using B-cell mitogens such as lipopolysaccharide, Epstein-Barr virus, staphylococcus protein A, or pokeweed mitogen. Three to 5 days later, the cells are treated with Colcemid to block cell division in metaphase, and the cells are recovered for analysis. Cells are treated with hypotonic solution to disrupt the nuclear membrane and then fixed in methanol and acetic acid, centrifuged, resuspended in fixative, and placed onto slides. Slides are stained with Giemsa or quinacrine to identify chromosome bands and are then photographed. Twenty-six metaphases are typically analyzed. Arrow denotes the extra chromosome 12.

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Figure 5.
Lymph node and bone marrow from a patient with chronic lymphocytic leukemia evolving to large B-cell lymphoma.Top left.Top right.Bottom left.Bottom right.

Low-power magnification of lymph node showing small-cell infiltration on the left and large cells on the right (hematoxylin and eosin; magnification 500). High-power magnification of the same lymph node showing small cells on the left and large cells on the right (magnification, 2500). Low-power magnification of bone marrow from the same patient showing small and large cells (hematoxylin and eosin; magnification, 500). High-power magnification of bone marrow showing predominantly small cells on the left and large cells on the right (hematoxylin and eosin; magnification, 2500).

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Grahic Jump Location
Figure 6.
Blood from a patient with prolymphocytic transformation showing small chronic lymphocytic leukemia cells and large prolymphocytes with nucleoli.

(Wright stain; magnification, 2500.).

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