Richter's Syndrome Presenting as Primary Central Nervous System Lymphoma Transformation of an Identical Clone
KATHERINE M. BAYLISS, M.D., BRIAN D. KUECK, M.D., CURTIS A. HANSON, M.D., WILLIAM G. MATTHAEUS, M.D., AND URIAS A. ALMAGRO, M.D.
DIFFUSE large cell, non-Hodgkin's lymphoma complicating chronic lymphocytic leukemia (CLL) was initially described by Richter in 192828 and has since been referred to as Richter's syndrome. Although the nature of the large cell lesion was initially unknown, subsequent advances in technology and our understanding of lymphoid ontogeny allow characterization of most of these lesions as Bcell lymphoproliferative malignancies. Previous investigations have not clarified whether the large cell lymphoma uniformly arises as a result of transformation of the small lymphocyte or if it is a second distinct malignancy. We report an unusual patient who presented with concurrent CLL and a central nervous system (CNS) large cell lymphoma. To our knowledge, Richter's syndrome confined to the CNS parenchyma has not been previously described. Evaluation of DNA content, phenotypic characterization, and molecular gene rearrangement studies were performed on peripheral blood and CNS tissue. The results of these studies suggest that at least some of the large cell lymphomas complicating CLL can evolve from the preexisting CLL clone.
Departments of Pathology and Medicine, Division of Hematology-Oncology, The Medical College of Wisconsin, Milwaukee, Wisconsin, and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
Report of a Case A 78-year-old white male was brought to the Zablocki Veteran's Administration Medical Center on July 6, 1988, for evaluation of confusion. Over the preceding four weeks, he began to experience progressive confusion, his appetite was poor, and he lost 12 pounds. In addition, he began having daily temperature elevations to 38.3-38.9 °C (101-102 °F) and urinary incontinence. The patient's medical history included hypertension and adult onset diabetes mellitus, which were managed with hydrochlorothiazide and chlorpropamide, respectively. On physical examination, he was a cachectic-appearing white man. He was alert but disoriented and unable to follow simple commands. No muscle weakness was appreciated. His blood pressure was 110/70 mmHg, with a pulse rate of 60 beats/minute and a temperature of 36.4 °C (97.6 °F). Results of the funduscopic examination were unremarkable. His neck was supple without lymphadenopathy. There were no palpable lymph nodes in the axillae or inguinal regions. Results of the cardiopulmonary examination were unremarkable. His abdomen was without organomegaly or masses. Laboratory evaluation revealed a hemoglobin of 158 g/L (15.8 g/dL), a hematocrit of 0.461 (46.1%), a white blood cell count of 13.9 X 109/ L with a differential cell count of 0.02 (2%) bands, 0.42 (42%) neutrophils, 0.53 (53%) lymphocytes, and 0.05 (5%) monocytes. The platelet count was 145 X 109/L. The electrolyte panel and calcium, phosphorous, magnesium, and liver function panel, with the exception of a lactate dehydrogenase level of 210 U/L (nL 100-190 U/L), were all normal. The urine showed only moderate glycosuria, and his chest x-ray was normal. Hospital Course The patient had a computed tomography (CT) scan of the head that demonstrated two ill-defined lesions with surrounding edema in the right temporal and the left frontal regions of the brain. He was given dexamethasone and had slight improvement in his mental status. On the fourth hospital day, he had a right fronto-temporal craniotomy with biopsy of a cerebral lesion that proved to be diffuse large cell lymphoma. Subsequent staging evaluation included a CT scan of the abdomen that was normal and a bone marrow biopsy that showed CLL. The patient's postoperative course was complicated by severe hypertension, the development of bilateral pulmonary infiltrates, and obtundation. Che-
Received March 6, 1989; received revised manuscript and accepted for publication June 19, 1989. Address reprint requests to Dr. Kueck: Milwaukee County Medical Complex, Department of Pathology, Box 152, 8700 West Wisconsin Avenue, Milwaukee, Wisconsin 53226.
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The development of a central nervous system (CNS) large cell lymphoma in a patient simultaneously diagnosed with chronic lymphocytic leukemia (CLL) is reported. Although differences in phenotypic expression were demonstrated in study of the peripheral blood and CNS disease, identical immunoglobulin gene rearrangements were identified, providing evidence for evolution of two morphologically distinct neoplasms from the same clone. Beyond histologic transformation, acquisition of an aneuploid cell population in the CNS tumor was demonstrated by analysis of DNA content. Isolated parenchymal involvement of the CNS by large cell transformation of CLL has not been previously described; its relationship to CNS lymphoma and Richter's syndrome are reviewed. (Key words: Richter's syndrome; CNS Lymphoma; CLL; DNA; Genotype; Immunophenotype) Am J Clin Pathol 1990;93:117-123
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motherapy and radiotherapy were discussed with the family, but these treatments were declined. The patient's condition continued to worsen, and he died on the 22nd hospital day. Autopsy revealed CNS lymphoma and bone marrowfindingsconsistent with CLL as well as severe bilateral pneumonia. The remainder of the evaluation was negative for lymphoma. Materials and Methods Histology
Immunologic Flow Cytometry Peripheral blood was collected in edetate (EDTA) Vacutainer® tubes. Whole blood lysis was performed in accordance with the instructions supplied by the Coulter Clone® whole blood lysis procedure. Briefly, appropriate concentrations of fluorescein isothiocyanate- or phycoerythrin-tagged antibodies were incubated with 100 /ih of venous blood. After the cells were washed with phosphatebuffered saline (PBS), the erythrocytes were lysed using Immunolyse®. Fixative was added after 90 seconds, and the cells were again washed and resuspended in PBS. A raw cell suspension was prepared from tissue by mechanical dissociation and filtration through nylon mesh and diluted with RPMI 1640 to approximately 4 X 109 cells/L. Appropriate concentrations of tagged antibodies were added to the cell suspension and incubated in a dark ice bath for 30 minutes. The cells were subsequently washed, then fixed with paraformaldehyde. Cells were analyzed with monoclonal antibodies to CD2 (Til), CD3 (T3), CD4 (T4), CD8 (T8), CD 19 (B4), CD20 (B1), CD 14 (My4), and 12 (Coulter Immunology, Hialeah, FL) and CD1 (Leu-9) and CD5 (Leu-1) (Becton-Dickinson, Mountain View, CA). F(ab')2 antibodies to immunoglobulin heavy and light chains (IgG, IgA, IgM, IgD, kappa, lambda) were supplied by Tago (Burlingame, CA.). Flow analysis was performed on the Coulter Epics C® flow cytometer. The lymphoid cell population was isolated with the use of forward-angle light scatter and 90-degree light scatter. Control preparations consisted of cells stained with an antibody of the same isotype as the test antibody. Five thousand events were collected. Single-parameter and dual-parameter histograms and scattergrams were analyzed for percentages of positive cells using Coulter software.
DNA Flow Cytometry DNA flow cytometry was performed on peripheral blood Ficoll-Hypaque® mononuclear layer preparations and the cell suspension prepared from the brain tissue. Cells at 1 X 109/L were centrifuged for 10 minutes at 1,000 RPM. The supernate was poured off and the pellet loosened by vortexing. The cells were then resuspended in 1.0 mL of 4 mmol/L citrate buffer, pH 7.8, containing 50 mg/L propidium iodine, 30 g/L PEG 8000, 0.2% (v/ v) Nonidet P-40®, and 5.0 X 104 RNase Kunitz units/L and placed in a 37 °C waterbath for 40 minutes; 1.0 mL of NaCl containing 50 mg/L propidium iodine, 30 g/L PEG 8000, and 0.2% (w/v) Nonidet P-40 were then added. The preparation was incubated at 4 °C for one hour. Flow cytometric (FCM) analysis was performed on an EPICS 742® flow cytometer (Coulter Electronics) using the 488-nm laser line operating at an output of 400 mW. Nuclei were gated on forward and 90-degree light scatter, and doublets were eliminated by bit mapping on peak versus integral red fluorescence signals. Approximately 15,000 nuclei were counted for each histogram. Go/Gi peak position was set at channel 50. Normal human lymphocytes were treated in the same fashion and used as an external diploid reference for each sample. Cell cycle analyses of FCM DNA histograms were calculated with Cytologic Software® (Coulter Electronics) using the "broadened rectangle" method. DNA Gene Rearrangement Studies The DNA used for gene rearrangement studies was extracted from snap-frozen cerebral tissue and peripheral blood according to standard procedures.23 Ten micrograms of DNA from each sample was digested with BamHl, EcoRl, or Hindlll (Bethesda Research Laboratory, Bethesda, MD) and size-fractioned by agarose gel (0.7% [w/v] electrophoresis); the DNA was then transferred from the gel to a nylon filter (Zetabind®, AMF Cumo) by Southern transfer technique in 20X SSC (IX = 150 mmol/L NaCl, 15 mmol/L NaCitrate, pH = 7.4).33 Filters were hybridized in 50% (v/v) formamide at 42 °C for 24-48 hours using DNA probes labeled with 32 P by the random primer method. 7 After hybridization, the filters were washed for 30 minutes to one hour in 0.1X SSC, 0.1% (w/v) sodium dodecyl sulfate (SDS) followed by autoradiography at - 7 0 °C. The Zetabind filters were reused by stripping the labeled probe from the filter with 0.5% (w/v) NaOH, 1.0% (w/v) SDS at 50 °C, placed back on radiographic film to confirm the absence of radioactive bands, and then rehybridized with another 32 Plabeled probe. Immunoglobulin gene probes used in this study consisted of a 6.0-kb BamHl/Hindlll fragment containing the joining region of the immunoglobulin heavy chain
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Portions of the cerebral mass were fixed in buffered formalin or B5, processed, embedded in paraffin, and cut in A-5-fim sections. A bone marrow trephine biopsy was fixed in Zenker's before decalcification and routine histologic processing. All tissue sections were stained with hematoxylin and eosin. In addition, Wright-Giemsastained bone marrow aspirate smears were prepared.
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gene (JH), the 2.5-kb EcoRl fragment from the constant region of the kappa light chain gene, and the 0.8 kb-EcoRl fragment from the constant region of the lambda light chain gene. Analysis of J H and kappa were performed with DNA digested with BamHl; lambda analysis used EcoR 1 -digested DNA. The T-cell receptor gene probe used was a 0.77-kb cDNA constant region probe of the beta chain gene (T-beta); BamHl-, Hindlll-, and EcoRl-digested DNA were separately evaluated with the T-beta probe. These probes have been described elsewhere.'' Results Pathology
chromatin and scanty cytoplasm were scattered in the background. Numerous mitotic figures were present. Histologic sections of the bone marrow biopsy showed diminished normal hematopoietic elements with multiple nonparatrabecular lymphoid aggregates composed of mature round lymphocytes. The aggregates were poorly defined, with lymphocytes infiltrating the adjacent interstitium (Fig. IB). Aspirate smears contained 0.45 (45%) mature lymphocytes; sheets of lymphocytes were also noted. There was no evidence of large cell lymphoma. Immunologic Cell Surface Markers The results of the cell surface marker studies by flow cytometry performed on both the cerebral tumor as well as the peripheral blood are provided in Table 1. Analysis of the peripheral blood showed a predominance of B-cells with IgM/D heavy chain specificity and strong Leu-1 positivity consistent with CLL. Light chain expression was not detected. Analysis of the brain lesion disclosed a predominance of B-cells with IgM-kappa monoclonality consistent with a malignant B-cell lymphoma.
m FIG. 1. A (left). Biopsy of the cerebral mass showing large lymphoid cells with vesicular chromatin frequently exhibiting multiple prominent nucleoli. Hematoxylin and eosin (X100). B (right). Bone marrow (clot section) showing an ill-defined aggregate of small round lymphocytes with dense nuclear chromatin. Hematoxylin and eosin (XI00).
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Histologic examination of the cerebral mass showed a diffusely infiltrating lymphoid malignancy. The infiltrate was composed predominantly of large cells with generally round nuclei, vesicular chromatin, prominent single to multiple nucleoli, dense nuclear membranes, and scanty to moderate amounts of amphophilic cytoplasm (Fig. \A). A few small round lymphocytes with moderately dense
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Table 1. Cell Surface Marker Results of the Peripheral Blood Lymphocytes (PB) and Central Nervous System (CNS) Lymphoma PB (%) 83 84 1 79 76 2 3 2 96 5 3 1 3 60 0 4
ND = not done. DNA Flow Cytometry Results of DNA flow cytometric analysis are depicted in Figure 2. Analysis of the peripheral blood showed a single Go/G] peak corresponding to a diploid cell population. Analysis of the brain lesion demonstrated two G 0 / G, peaks. The major cell population appeared diploid, whereas the second near-diploid aneuploid peak (DNA index 1.09) accounted for 6.5% of the cells. Genotyping Studies DNA from the CNS tumor and the peripheral blood lymphocytes showed identical rearrangements with the J H and kappa gene probes (Fig. 3). Rearranged bands were of strong intensity, suggesting that most cells in both specimens contained clonal rearrangements. Analysis with lambda and T-beta gene probes showed germline configuration. Discussion Most lymphomas found in the CNS are secondary, or metastatic, occurring during the course of disease in 5-11.5% of patients with non-Hodgkin's lymphomas. 1317,18,22 Parenchymal involvement of the CNS accounts for less than one-third of these cases, most involving the leptomeninges or perivascular spaces. I314 Primary CNS lymphoma has been reported to account for 1 -2% of all malignant non-Hodgkin's lymphomas, typically presenting as deep seeded expanding masses; up to 45% are multicentric at the time of diagnosis. The patient presented in this study does not accurately fit the definition of either primary or secondary CNS lymphoma and may more precisely be described as having
Richter's syndrome, presenting in and confined to the brain. Richter's syndrome, estimated to occur in 3-15% of patients with CLL, 114 is typically heralded by a fairly abrupt deterioration of the patient's chronic state. Lymph node or bone marrow infiltration at the time of large cell transformation is typical. Extranodal involvement is not uncommon, although disease localized to a single extranodal site is unusual1'9'20,35; this has been reported to occur in the gastrointestinal tract.4 In patients experiencing large cell transformation, the mean interval of time from the diagnosis of CLL to a large cell non-Hodgkin's lymphoma has been reported as two to four years. 112 Occasionally, the diagnosis is made concomitantly. 16 No identifiable distinguishing features of CLL have proved useful in predicting transformation. In contrast to the acute leukemias, CNS involvement by CLL is uncommon, with only a few cases reported in the literature.1019 Similarly, Richter's syndrome involving the CNS is rare and, on review of the literature, Richter's syndrome confined to the brain parenchyma has not been reported. A recent case report16 describes a patient with simultaneous occurrence of CLL, extranodal large cell non-Hodgkin's lymphoma, and malignant meningeal involvement. The clonal nature of cells isolated from peripheral blood, cerebrospinal fluid, and extranodal tissue was substantiated by cell surface marker studies. The case presently reported, however, stands in distinction because the CNS lesion was parenchymal and occurred as isolated extranodal involvement; no additional sites of transformation were identified at autopsy. It has been established that patients with CLL have an increased risk for development of second neoplasms,3 yet there appears to be a disproportionately high occurrence of large cell lymphoma. Continued controversy exists as to whether the large cell component of Richter's syndrome emerges from the preexisting CLL or represents a second de novo neoplasm. Evidence for both theories has been provided by cytogenetic studies, immunophenotypic analysis, and, more recently, genetic studies using Southern blotting techniques. Previous reports of karyotypic analysis have suggested clonal evolution. Nowell and associates25 demonstrated similar marker chromosomes in a patient with T-CLL in whom a large cell lymphoma subsequently developed. The marker chromosome appeared over time on sequential monitoring of the patient's CLL and was found as a repetitive feature on analysis of the large cell lymphoma. The karyotypic study reported by Fitzgerald and colleagues8 in a patient with more typical B-CLL and large cell transformation demonstrated clonal abnormalities in the large cell neoplasm in contrast to the patient's peripheral blood, which was diploid. It is not clear, however, that the study of the patient's peripheral blood truly reflected the genetic makeup of the CLL lymphocytes.
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Bl B4 IgG IgD IgM IgA Kappa Lambda Leu-1 Til T3 T8 T4 12 My4 Leu-9
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Some studies using immunophenotyping techniques have demonstrated the cells of CLL and Richter's syndrome to express similar heavy chains of the immunoglobulin molecule but different light chains,24'31,34'36 suggesting two unrelated lymphoid neoplasms. An equal number of reports have shown identical surface immunoglobulin on lymphocytes of CLL and large cell component of Richter's syndrome 2 ' 5 ' 216 ; these reports infer the Richter's syndrome to originate from the same clone of cells as the CLL, or histologic transformation of an identical clone. It has been shown, however, that lymphomas of different histologic subtypes but identical immunophenotype within the same patient can have differ-
ent clonal immunoglobulin gene rearrangements, thereby providing evidence of different clonal origin.32 From these reports it is clear that the sole use of surface immunoglobulin heavy and light chain staining is least likely to answer the question of clonal relationship. Monoclonal antiidiotype antibodies would more likely provide a definitive answer; such studies are expensive and time consuming and, to our knowledge, have yet to be reported in CLL. The more recent technology of Southern blotting to detect immunoglobulin gene rearrangements provides more definitive answers. Although in the case reported here cell surface marker analysis failed to show identical
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FIG. 2. A (upper). Analysis of the patient's peripheral blood lymphocytes showing a diploid (2C) pattern. A prominent G0/G, peak is at channel 50 (CV = 1.96); a small G2/M peak is seen at channel 100. B (lower). Analysis of the CNS lymphoma showing a prominent diploid peak at channel 50 (CV = 2.1) and a second aneuploid peak (arrow) with DNA index of 1.09 (CV = 2.3). The G2/M peaks of both diploid and aneuploid cell populations are seen as a broad peak near channel 100.
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Bam HI - J H 1
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C
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FlG. 3. Southern blot hybridization analysis of JH and kappa immunoglobulin genes in DNA extracted from the patient's peripheral blood (lane I) and CNS tissue (lane 2) using Bam H l digests. Arrows denote rearranged bands with germline bands denoted by dashes at 17 kb and 12 kb with JH and Kappa genes, respectively. C denotes control.
transformation. Relatively few cases of Richter's syndrome analyzed by gene rearrangement studies or quantitative DNA flow cytometry have been reported. Undoubtedly, continued use of these relatively new tools will make significant contributions to our understanding of the relationship of the two lymphoid lesions that constitute this syndrome, as well as advance our general understanding of the biologic behavior of lymphoid malignancies. Acknowledgments. The authors acknowledge Patrick W. McFadden for his contributions to the DNA studies. They also thank Elizabeth Larson for her assistance in preparation of the manuscript. References 1. Armitage JO, Dick FR, Corder MP. Diffuse histiocytic lymphoma complicating chronic lymphocytic leukemia. Cancer 1978;41:422427. 2. Baumann MA, Libnoch JA, Patrick CW, Choi H, Keller RH. Prolonged survival in Richter syndrome with subsequent reemergence of CLL: a case report including serial cell-surface phenotypic analysis. Am J Hematol 1985;20:67-72. 3. Berg JW. The incidence of multiple primary cancers. I. Development of further cancers in patients with lymphomas, leukemias and myeloma. JNCI 1967;38:741-752. 4. Brousse N, Solal-Celigny P, Herrera A, et al. Gastrointestinal Richter's syndrome. Hum Pathol 1985;16:854-857. 5. Delsol G, Laurent G, Kuhlein E, Familiades J, Rigal F, Pris J. Richter's syndrome. Evidence for the clonal origin of the two proliferations. Am J Clin Pathol. 1981;76:308-315. 6. Diamond LW, Nathwani BN, Rappaport H. Flow cytometry in the diagnosis and classification of malignant lymphoma and leukemia. Cancer 1982;50:1122-1135. 7. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13. 8. Fitzgerald PH, McEwan CM, Hamer JW, Beard MEJ. Richter's syndrome with identification of marker chromosomes. Cancer 1980;46:135-138. 9. Foucar K, Rydell RE. Richter's syndrome in chronic lymphocytic leukemia. Cancer 1980;46:118-134. 10. Getaz EP, Miller GJ. Spinal cord involvement in chronic lymphocytic leukemia. Cancer 1979;43:1858-1861. 11. Hanson CA, Frizzera G, Patton DF, et al. Clonal rearrangement for immunoglobulin and T-cell receptor genes in systemic Castle-
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phenotypic expression, DNA analysis by Southern blotting clearly established a clonal relationship. This contrasts with the reports of Ostrowski and associates26 and vanDongen and associates.36 Using gene probes, these reports describe different gene rearrangements, suggesting that the large cell lymphoma arose from a different clone than the CLL. We must emphasize the necessity of using both immunoglobulin heavy chain and light chain gene probes when evaluating cases of Richter's syndrome to determine clonal origin. Immunoglobulin heavy chain gene studies alone are not conclusive because investigations of postrearrangement deletions of immunoglobulin heavy chain genes can occur32; these would appear as unique rearranged bands, suggesting separate clones. Such deletions have not been reported with immunoglobulin light chain genes. In addition, immunoglobulin heavy chain switching is a well-recognized phenomenon that may also give rise to unique sized rearranged bands by Southern blotting methods. Analysis of the DNA content by flow cytometry showed the presence of a small aneuploid cell population in the large cell lesion, unlike the singularly diploid small cell component. This is consistent with several studies that demonstrate a significantly higher incidence of aneuploidy in histologically aggressive B-cell lymphoid lesions {i.e., corresponding to the intermediate and high-grade lymphomas of the Working Formulation).6,15-21-27-2930 Indeed, some patients shown to have histologic transformation of low-grade lymphoma to high-grade lesions have demonstrated acquisition of an aneuploid cell population.30 Similarly, in this case of Richter's transformation this finding suggests that a chromosomal alteration accompanied the morphologic transformation of the low-grade lesion. We have reported a patient with a very unusual presentation of Richter's syndrome. A variety of techniques were used to analyze and characterize the nature of this
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24. McDonnell JM, Beschorner WE, Staal SP, Spivak JL, Mann RB. Richter's syndrome with two different B-cell clones. Cancer. 1986;58:2031-2037. 25. Nowell P, Finan J, Glover D, Guerry D. Cytogenetic evidence for the clonal nature of Richter's syndrome. Blood 1981;58:183-186. 26. Ostrowski M, Minden M, Wang C, Bailey D. Immunoperoxidase and gene probe analysis of a case of Richter's syndrome. Am J Clin Pathol 1989;91:215-221. 27. Palutke M, Schnitzer B, Dresner D, et al. Comparison of morphologic features and mitotic rate to cytometrically determined DNA content of poorly differentiated lymphocytic lymphomas. Ann NY AcadSci 1986;468:178-194. 28. Richter MN. Generalized reticular cell sarcoma of the lymph nodes associated with lymphocytic leukemia. Am J Pathol 1928,4:285299. 29. Shackney SE. The use offlowcytometry in the diagnosis and biological characterization of non-Hodgkin's lymphomas. Ann NY AcadSci 1986;468:171-177. 30. Shackney SE, Levine AM, Fisher RI, et al. The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases. J Clin Invest 1984;73:1201-1214. 31. Sheibani K, Nathwani BN, Winberg CD, Scott EP, Teplitz RR, Rappaport H. Small lymphocytic lymphoma. Morphologic and immunologic progression. Am J Clin Pathol 1985;84:237-243. 32. Siegelman MH, Cleary ML, Warnke R, Sklar J. Frequent biclonality and Ig gene alterations among B cell lymphomas that show multiple histologic forms. J Exp Med 1985;161:850-863. 33. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-517. 34. Splinter TAW, Noorloos AB, Van Heerde P. CLL and diffuse histiocytic lymphoma in one patient: clonal proliferation of two different B cells. Scand J Haematol 1978;20:29-36. 35. Trump DL, Mann RB, Phelps R, Roberts H, Conley CL. Richter's syndrome: diffuse histiocytic lymphoma in patients with chronic lymphocytic leukemia. A report offivecases and review of the literature. Am J Med 1980;68:539-548. 36. van Dongen JJM, Hooijkaas H, Michiels JJ, et al. Richter's syndrome with different immunoglobulin light chains and different heavy chain gene rearrangements. Blood 1984;64:571-575.
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man's disease: association with Epstein-Barr virus. Am J Pathol 1988;I31:84. 12. Harousseau JL, Flandrin G, Tricot G, Brouet JC, Seligmann M, Bernard J. Malignant lymphoma supervening in chronic lymphocytic leukemia and related disorders. Richter's syndrome: a study of 25 cases. Cancer 1981 ;48:1302-1308. 13. Herman TS, Hammond N, Jones SE, Butler JJ, Byrne GE, McKelvey EM. Involvement of the central nervous system by non-Hodgkin's lymphoma. The Southwest Oncology Group experience. Cancer 1979;43:390-397. 14. JafTe ES. Surgical Pathology of the lymph nodes and related organs. Philadelphia: WB Saunders, 1985. 15. Juneja SK, Cooper IA, Hodgson GS, et al. DNA ploidy patterns and cytokinetics of non-Hodgkin's lymphoma. J Clin Pathol 1986;39:987-992. 16. Lane PK, Townsend RM, Beckstead JH, Corash L. Central nervous system involvement in a patient with chronic lymphocytic leukemia and non-Hodgkin's lymphoma (Richter's syndrome), with concordant cell surface immunoglobulin isotypic and immunophenotypic markers. Am J Clin Pathol 1988;89:254-259. 17. Law IP, Dick FR, Blom J, Bergevin PR. Involvement of the central nervous system in non-Hodgkin's lymphoma. Cancer 1975;36: 225-231. 18. Levitt U, Dawson DM, Rosenthal DS, Moloney WC. CNS involvement in non-Hodgkin's lymphomas. Cancer 1980;45:545-552. 19. Liepman MK, Votaw ML. Meningeal leukemia complicating chronic lymphocytic leukemia. Cancer 1981;47:2482-2484. 20. Long JC, Aisenberg AC. Richter's syndrome. A terminal complication of chronic lymphocytic leukemia with distinct clinicopathologic features. Am J Clin Pathol 1975;63:786-795. 21. Macartney JC, Camplejohn RS, Adler J, Stone MG, Powell G. Prognostic importance of DNA flow cytometry in non-Hodgkin's lymphomas. J Clin Pathol 1986;39:542-546. 22. Mackintosh FR, Colby TV, Podolsky WJ, et al. Central nervous system involvement in non-Hodgkin's lymphoma: an analysis of 105 cases. Cancer 1982;49:586-595. 23. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982.
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KATHERINE M. BAYLISS, M.D., BRIAN D. KUECK, M.D., CURTIS A. HANSON, M.D., WILLIAM G. MATTHAEUS, M.D., AND URIAS A. ALMAGRO, M.D.
DIFFUSE large cell, non-Hodgkin's lymphoma complicating chronic lymphocytic leukemia (CLL) was initially described by Richter in 192828 and has since been referred to as Richter's syndrome. Although the nature of the large cell lesion was initially unknown, subsequent advances in technology and our understanding of lymphoid ontogeny allow characterization of most of these lesions as Bcell lymphoproliferative malignancies. Previous investigations have not clarified whether the large cell lymphoma uniformly arises as a result of transformation of the small lymphocyte or if it is a second distinct malignancy. We report an unusual patient who presented with concurrent CLL and a central nervous system (CNS) large cell lymphoma. To our knowledge, Richter's syndrome confined to the CNS parenchyma has not been previously described. Evaluation of DNA content, phenotypic characterization, and molecular gene rearrangement studies were performed on peripheral blood and CNS tissue. The results of these studies suggest that at least some of the large cell lymphomas complicating CLL can evolve from the preexisting CLL clone.
Departments of Pathology and Medicine, Division of Hematology-Oncology, The Medical College of Wisconsin, Milwaukee, Wisconsin, and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
Report of a Case A 78-year-old white male was brought to the Zablocki Veteran's Administration Medical Center on July 6, 1988, for evaluation of confusion. Over the preceding four weeks, he began to experience progressive confusion, his appetite was poor, and he lost 12 pounds. In addition, he began having daily temperature elevations to 38.3-38.9 °C (101-102 °F) and urinary incontinence. The patient's medical history included hypertension and adult onset diabetes mellitus, which were managed with hydrochlorothiazide and chlorpropamide, respectively. On physical examination, he was a cachectic-appearing white man. He was alert but disoriented and unable to follow simple commands. No muscle weakness was appreciated. His blood pressure was 110/70 mmHg, with a pulse rate of 60 beats/minute and a temperature of 36.4 °C (97.6 °F). Results of the funduscopic examination were unremarkable. His neck was supple without lymphadenopathy. There were no palpable lymph nodes in the axillae or inguinal regions. Results of the cardiopulmonary examination were unremarkable. His abdomen was without organomegaly or masses. Laboratory evaluation revealed a hemoglobin of 158 g/L (15.8 g/dL), a hematocrit of 0.461 (46.1%), a white blood cell count of 13.9 X 109/ L with a differential cell count of 0.02 (2%) bands, 0.42 (42%) neutrophils, 0.53 (53%) lymphocytes, and 0.05 (5%) monocytes. The platelet count was 145 X 109/L. The electrolyte panel and calcium, phosphorous, magnesium, and liver function panel, with the exception of a lactate dehydrogenase level of 210 U/L (nL 100-190 U/L), were all normal. The urine showed only moderate glycosuria, and his chest x-ray was normal. Hospital Course The patient had a computed tomography (CT) scan of the head that demonstrated two ill-defined lesions with surrounding edema in the right temporal and the left frontal regions of the brain. He was given dexamethasone and had slight improvement in his mental status. On the fourth hospital day, he had a right fronto-temporal craniotomy with biopsy of a cerebral lesion that proved to be diffuse large cell lymphoma. Subsequent staging evaluation included a CT scan of the abdomen that was normal and a bone marrow biopsy that showed CLL. The patient's postoperative course was complicated by severe hypertension, the development of bilateral pulmonary infiltrates, and obtundation. Che-
Received March 6, 1989; received revised manuscript and accepted for publication June 19, 1989. Address reprint requests to Dr. Kueck: Milwaukee County Medical Complex, Department of Pathology, Box 152, 8700 West Wisconsin Avenue, Milwaukee, Wisconsin 53226.
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The development of a central nervous system (CNS) large cell lymphoma in a patient simultaneously diagnosed with chronic lymphocytic leukemia (CLL) is reported. Although differences in phenotypic expression were demonstrated in study of the peripheral blood and CNS disease, identical immunoglobulin gene rearrangements were identified, providing evidence for evolution of two morphologically distinct neoplasms from the same clone. Beyond histologic transformation, acquisition of an aneuploid cell population in the CNS tumor was demonstrated by analysis of DNA content. Isolated parenchymal involvement of the CNS by large cell transformation of CLL has not been previously described; its relationship to CNS lymphoma and Richter's syndrome are reviewed. (Key words: Richter's syndrome; CNS Lymphoma; CLL; DNA; Genotype; Immunophenotype) Am J Clin Pathol 1990;93:117-123
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motherapy and radiotherapy were discussed with the family, but these treatments were declined. The patient's condition continued to worsen, and he died on the 22nd hospital day. Autopsy revealed CNS lymphoma and bone marrowfindingsconsistent with CLL as well as severe bilateral pneumonia. The remainder of the evaluation was negative for lymphoma. Materials and Methods Histology
Immunologic Flow Cytometry Peripheral blood was collected in edetate (EDTA) Vacutainer® tubes. Whole blood lysis was performed in accordance with the instructions supplied by the Coulter Clone® whole blood lysis procedure. Briefly, appropriate concentrations of fluorescein isothiocyanate- or phycoerythrin-tagged antibodies were incubated with 100 /ih of venous blood. After the cells were washed with phosphatebuffered saline (PBS), the erythrocytes were lysed using Immunolyse®. Fixative was added after 90 seconds, and the cells were again washed and resuspended in PBS. A raw cell suspension was prepared from tissue by mechanical dissociation and filtration through nylon mesh and diluted with RPMI 1640 to approximately 4 X 109 cells/L. Appropriate concentrations of tagged antibodies were added to the cell suspension and incubated in a dark ice bath for 30 minutes. The cells were subsequently washed, then fixed with paraformaldehyde. Cells were analyzed with monoclonal antibodies to CD2 (Til), CD3 (T3), CD4 (T4), CD8 (T8), CD 19 (B4), CD20 (B1), CD 14 (My4), and 12 (Coulter Immunology, Hialeah, FL) and CD1 (Leu-9) and CD5 (Leu-1) (Becton-Dickinson, Mountain View, CA). F(ab')2 antibodies to immunoglobulin heavy and light chains (IgG, IgA, IgM, IgD, kappa, lambda) were supplied by Tago (Burlingame, CA.). Flow analysis was performed on the Coulter Epics C® flow cytometer. The lymphoid cell population was isolated with the use of forward-angle light scatter and 90-degree light scatter. Control preparations consisted of cells stained with an antibody of the same isotype as the test antibody. Five thousand events were collected. Single-parameter and dual-parameter histograms and scattergrams were analyzed for percentages of positive cells using Coulter software.
DNA Flow Cytometry DNA flow cytometry was performed on peripheral blood Ficoll-Hypaque® mononuclear layer preparations and the cell suspension prepared from the brain tissue. Cells at 1 X 109/L were centrifuged for 10 minutes at 1,000 RPM. The supernate was poured off and the pellet loosened by vortexing. The cells were then resuspended in 1.0 mL of 4 mmol/L citrate buffer, pH 7.8, containing 50 mg/L propidium iodine, 30 g/L PEG 8000, 0.2% (v/ v) Nonidet P-40®, and 5.0 X 104 RNase Kunitz units/L and placed in a 37 °C waterbath for 40 minutes; 1.0 mL of NaCl containing 50 mg/L propidium iodine, 30 g/L PEG 8000, and 0.2% (w/v) Nonidet P-40 were then added. The preparation was incubated at 4 °C for one hour. Flow cytometric (FCM) analysis was performed on an EPICS 742® flow cytometer (Coulter Electronics) using the 488-nm laser line operating at an output of 400 mW. Nuclei were gated on forward and 90-degree light scatter, and doublets were eliminated by bit mapping on peak versus integral red fluorescence signals. Approximately 15,000 nuclei were counted for each histogram. Go/Gi peak position was set at channel 50. Normal human lymphocytes were treated in the same fashion and used as an external diploid reference for each sample. Cell cycle analyses of FCM DNA histograms were calculated with Cytologic Software® (Coulter Electronics) using the "broadened rectangle" method. DNA Gene Rearrangement Studies The DNA used for gene rearrangement studies was extracted from snap-frozen cerebral tissue and peripheral blood according to standard procedures.23 Ten micrograms of DNA from each sample was digested with BamHl, EcoRl, or Hindlll (Bethesda Research Laboratory, Bethesda, MD) and size-fractioned by agarose gel (0.7% [w/v] electrophoresis); the DNA was then transferred from the gel to a nylon filter (Zetabind®, AMF Cumo) by Southern transfer technique in 20X SSC (IX = 150 mmol/L NaCl, 15 mmol/L NaCitrate, pH = 7.4).33 Filters were hybridized in 50% (v/v) formamide at 42 °C for 24-48 hours using DNA probes labeled with 32 P by the random primer method. 7 After hybridization, the filters were washed for 30 minutes to one hour in 0.1X SSC, 0.1% (w/v) sodium dodecyl sulfate (SDS) followed by autoradiography at - 7 0 °C. The Zetabind filters were reused by stripping the labeled probe from the filter with 0.5% (w/v) NaOH, 1.0% (w/v) SDS at 50 °C, placed back on radiographic film to confirm the absence of radioactive bands, and then rehybridized with another 32 Plabeled probe. Immunoglobulin gene probes used in this study consisted of a 6.0-kb BamHl/Hindlll fragment containing the joining region of the immunoglobulin heavy chain
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Portions of the cerebral mass were fixed in buffered formalin or B5, processed, embedded in paraffin, and cut in A-5-fim sections. A bone marrow trephine biopsy was fixed in Zenker's before decalcification and routine histologic processing. All tissue sections were stained with hematoxylin and eosin. In addition, Wright-Giemsastained bone marrow aspirate smears were prepared.
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gene (JH), the 2.5-kb EcoRl fragment from the constant region of the kappa light chain gene, and the 0.8 kb-EcoRl fragment from the constant region of the lambda light chain gene. Analysis of J H and kappa were performed with DNA digested with BamHl; lambda analysis used EcoR 1 -digested DNA. The T-cell receptor gene probe used was a 0.77-kb cDNA constant region probe of the beta chain gene (T-beta); BamHl-, Hindlll-, and EcoRl-digested DNA were separately evaluated with the T-beta probe. These probes have been described elsewhere.'' Results Pathology
chromatin and scanty cytoplasm were scattered in the background. Numerous mitotic figures were present. Histologic sections of the bone marrow biopsy showed diminished normal hematopoietic elements with multiple nonparatrabecular lymphoid aggregates composed of mature round lymphocytes. The aggregates were poorly defined, with lymphocytes infiltrating the adjacent interstitium (Fig. IB). Aspirate smears contained 0.45 (45%) mature lymphocytes; sheets of lymphocytes were also noted. There was no evidence of large cell lymphoma. Immunologic Cell Surface Markers The results of the cell surface marker studies by flow cytometry performed on both the cerebral tumor as well as the peripheral blood are provided in Table 1. Analysis of the peripheral blood showed a predominance of B-cells with IgM/D heavy chain specificity and strong Leu-1 positivity consistent with CLL. Light chain expression was not detected. Analysis of the brain lesion disclosed a predominance of B-cells with IgM-kappa monoclonality consistent with a malignant B-cell lymphoma.
m FIG. 1. A (left). Biopsy of the cerebral mass showing large lymphoid cells with vesicular chromatin frequently exhibiting multiple prominent nucleoli. Hematoxylin and eosin (X100). B (right). Bone marrow (clot section) showing an ill-defined aggregate of small round lymphocytes with dense nuclear chromatin. Hematoxylin and eosin (XI00).
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Histologic examination of the cerebral mass showed a diffusely infiltrating lymphoid malignancy. The infiltrate was composed predominantly of large cells with generally round nuclei, vesicular chromatin, prominent single to multiple nucleoli, dense nuclear membranes, and scanty to moderate amounts of amphophilic cytoplasm (Fig. \A). A few small round lymphocytes with moderately dense
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Table 1. Cell Surface Marker Results of the Peripheral Blood Lymphocytes (PB) and Central Nervous System (CNS) Lymphoma PB (%) 83 84 1 79 76 2 3 2 96 5 3 1 3 60 0 4
ND = not done. DNA Flow Cytometry Results of DNA flow cytometric analysis are depicted in Figure 2. Analysis of the peripheral blood showed a single Go/G] peak corresponding to a diploid cell population. Analysis of the brain lesion demonstrated two G 0 / G, peaks. The major cell population appeared diploid, whereas the second near-diploid aneuploid peak (DNA index 1.09) accounted for 6.5% of the cells. Genotyping Studies DNA from the CNS tumor and the peripheral blood lymphocytes showed identical rearrangements with the J H and kappa gene probes (Fig. 3). Rearranged bands were of strong intensity, suggesting that most cells in both specimens contained clonal rearrangements. Analysis with lambda and T-beta gene probes showed germline configuration. Discussion Most lymphomas found in the CNS are secondary, or metastatic, occurring during the course of disease in 5-11.5% of patients with non-Hodgkin's lymphomas. 1317,18,22 Parenchymal involvement of the CNS accounts for less than one-third of these cases, most involving the leptomeninges or perivascular spaces. I314 Primary CNS lymphoma has been reported to account for 1 -2% of all malignant non-Hodgkin's lymphomas, typically presenting as deep seeded expanding masses; up to 45% are multicentric at the time of diagnosis. The patient presented in this study does not accurately fit the definition of either primary or secondary CNS lymphoma and may more precisely be described as having
Richter's syndrome, presenting in and confined to the brain. Richter's syndrome, estimated to occur in 3-15% of patients with CLL, 114 is typically heralded by a fairly abrupt deterioration of the patient's chronic state. Lymph node or bone marrow infiltration at the time of large cell transformation is typical. Extranodal involvement is not uncommon, although disease localized to a single extranodal site is unusual1'9'20,35; this has been reported to occur in the gastrointestinal tract.4 In patients experiencing large cell transformation, the mean interval of time from the diagnosis of CLL to a large cell non-Hodgkin's lymphoma has been reported as two to four years. 112 Occasionally, the diagnosis is made concomitantly. 16 No identifiable distinguishing features of CLL have proved useful in predicting transformation. In contrast to the acute leukemias, CNS involvement by CLL is uncommon, with only a few cases reported in the literature.1019 Similarly, Richter's syndrome involving the CNS is rare and, on review of the literature, Richter's syndrome confined to the brain parenchyma has not been reported. A recent case report16 describes a patient with simultaneous occurrence of CLL, extranodal large cell non-Hodgkin's lymphoma, and malignant meningeal involvement. The clonal nature of cells isolated from peripheral blood, cerebrospinal fluid, and extranodal tissue was substantiated by cell surface marker studies. The case presently reported, however, stands in distinction because the CNS lesion was parenchymal and occurred as isolated extranodal involvement; no additional sites of transformation were identified at autopsy. It has been established that patients with CLL have an increased risk for development of second neoplasms,3 yet there appears to be a disproportionately high occurrence of large cell lymphoma. Continued controversy exists as to whether the large cell component of Richter's syndrome emerges from the preexisting CLL or represents a second de novo neoplasm. Evidence for both theories has been provided by cytogenetic studies, immunophenotypic analysis, and, more recently, genetic studies using Southern blotting techniques. Previous reports of karyotypic analysis have suggested clonal evolution. Nowell and associates25 demonstrated similar marker chromosomes in a patient with T-CLL in whom a large cell lymphoma subsequently developed. The marker chromosome appeared over time on sequential monitoring of the patient's CLL and was found as a repetitive feature on analysis of the large cell lymphoma. The karyotypic study reported by Fitzgerald and colleagues8 in a patient with more typical B-CLL and large cell transformation demonstrated clonal abnormalities in the large cell neoplasm in contrast to the patient's peripheral blood, which was diploid. It is not clear, however, that the study of the patient's peripheral blood truly reflected the genetic makeup of the CLL lymphocytes.
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Bl B4 IgG IgD IgM IgA Kappa Lambda Leu-1 Til T3 T8 T4 12 My4 Leu-9
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Some studies using immunophenotyping techniques have demonstrated the cells of CLL and Richter's syndrome to express similar heavy chains of the immunoglobulin molecule but different light chains,24'31,34'36 suggesting two unrelated lymphoid neoplasms. An equal number of reports have shown identical surface immunoglobulin on lymphocytes of CLL and large cell component of Richter's syndrome 2 ' 5 ' 216 ; these reports infer the Richter's syndrome to originate from the same clone of cells as the CLL, or histologic transformation of an identical clone. It has been shown, however, that lymphomas of different histologic subtypes but identical immunophenotype within the same patient can have differ-
ent clonal immunoglobulin gene rearrangements, thereby providing evidence of different clonal origin.32 From these reports it is clear that the sole use of surface immunoglobulin heavy and light chain staining is least likely to answer the question of clonal relationship. Monoclonal antiidiotype antibodies would more likely provide a definitive answer; such studies are expensive and time consuming and, to our knowledge, have yet to be reported in CLL. The more recent technology of Southern blotting to detect immunoglobulin gene rearrangements provides more definitive answers. Although in the case reported here cell surface marker analysis failed to show identical
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FIG. 2. A (upper). Analysis of the patient's peripheral blood lymphocytes showing a diploid (2C) pattern. A prominent G0/G, peak is at channel 50 (CV = 1.96); a small G2/M peak is seen at channel 100. B (lower). Analysis of the CNS lymphoma showing a prominent diploid peak at channel 50 (CV = 2.1) and a second aneuploid peak (arrow) with DNA index of 1.09 (CV = 2.3). The G2/M peaks of both diploid and aneuploid cell populations are seen as a broad peak near channel 100.
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C
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FlG. 3. Southern blot hybridization analysis of JH and kappa immunoglobulin genes in DNA extracted from the patient's peripheral blood (lane I) and CNS tissue (lane 2) using Bam H l digests. Arrows denote rearranged bands with germline bands denoted by dashes at 17 kb and 12 kb with JH and Kappa genes, respectively. C denotes control.
transformation. Relatively few cases of Richter's syndrome analyzed by gene rearrangement studies or quantitative DNA flow cytometry have been reported. Undoubtedly, continued use of these relatively new tools will make significant contributions to our understanding of the relationship of the two lymphoid lesions that constitute this syndrome, as well as advance our general understanding of the biologic behavior of lymphoid malignancies. Acknowledgments. The authors acknowledge Patrick W. McFadden for his contributions to the DNA studies. They also thank Elizabeth Larson for her assistance in preparation of the manuscript. References 1. Armitage JO, Dick FR, Corder MP. Diffuse histiocytic lymphoma complicating chronic lymphocytic leukemia. Cancer 1978;41:422427. 2. Baumann MA, Libnoch JA, Patrick CW, Choi H, Keller RH. Prolonged survival in Richter syndrome with subsequent reemergence of CLL: a case report including serial cell-surface phenotypic analysis. Am J Hematol 1985;20:67-72. 3. Berg JW. The incidence of multiple primary cancers. I. Development of further cancers in patients with lymphomas, leukemias and myeloma. JNCI 1967;38:741-752. 4. Brousse N, Solal-Celigny P, Herrera A, et al. Gastrointestinal Richter's syndrome. Hum Pathol 1985;16:854-857. 5. Delsol G, Laurent G, Kuhlein E, Familiades J, Rigal F, Pris J. Richter's syndrome. Evidence for the clonal origin of the two proliferations. Am J Clin Pathol. 1981;76:308-315. 6. Diamond LW, Nathwani BN, Rappaport H. Flow cytometry in the diagnosis and classification of malignant lymphoma and leukemia. Cancer 1982;50:1122-1135. 7. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13. 8. Fitzgerald PH, McEwan CM, Hamer JW, Beard MEJ. Richter's syndrome with identification of marker chromosomes. Cancer 1980;46:135-138. 9. Foucar K, Rydell RE. Richter's syndrome in chronic lymphocytic leukemia. Cancer 1980;46:118-134. 10. Getaz EP, Miller GJ. Spinal cord involvement in chronic lymphocytic leukemia. Cancer 1979;43:1858-1861. 11. Hanson CA, Frizzera G, Patton DF, et al. Clonal rearrangement for immunoglobulin and T-cell receptor genes in systemic Castle-
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phenotypic expression, DNA analysis by Southern blotting clearly established a clonal relationship. This contrasts with the reports of Ostrowski and associates26 and vanDongen and associates.36 Using gene probes, these reports describe different gene rearrangements, suggesting that the large cell lymphoma arose from a different clone than the CLL. We must emphasize the necessity of using both immunoglobulin heavy chain and light chain gene probes when evaluating cases of Richter's syndrome to determine clonal origin. Immunoglobulin heavy chain gene studies alone are not conclusive because investigations of postrearrangement deletions of immunoglobulin heavy chain genes can occur32; these would appear as unique rearranged bands, suggesting separate clones. Such deletions have not been reported with immunoglobulin light chain genes. In addition, immunoglobulin heavy chain switching is a well-recognized phenomenon that may also give rise to unique sized rearranged bands by Southern blotting methods. Analysis of the DNA content by flow cytometry showed the presence of a small aneuploid cell population in the large cell lesion, unlike the singularly diploid small cell component. This is consistent with several studies that demonstrate a significantly higher incidence of aneuploidy in histologically aggressive B-cell lymphoid lesions {i.e., corresponding to the intermediate and high-grade lymphomas of the Working Formulation).6,15-21-27-2930 Indeed, some patients shown to have histologic transformation of low-grade lymphoma to high-grade lesions have demonstrated acquisition of an aneuploid cell population.30 Similarly, in this case of Richter's transformation this finding suggests that a chromosomal alteration accompanied the morphologic transformation of the low-grade lesion. We have reported a patient with a very unusual presentation of Richter's syndrome. A variety of techniques were used to analyze and characterize the nature of this
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24. McDonnell JM, Beschorner WE, Staal SP, Spivak JL, Mann RB. Richter's syndrome with two different B-cell clones. Cancer. 1986;58:2031-2037. 25. Nowell P, Finan J, Glover D, Guerry D. Cytogenetic evidence for the clonal nature of Richter's syndrome. Blood 1981;58:183-186. 26. Ostrowski M, Minden M, Wang C, Bailey D. Immunoperoxidase and gene probe analysis of a case of Richter's syndrome. Am J Clin Pathol 1989;91:215-221. 27. Palutke M, Schnitzer B, Dresner D, et al. Comparison of morphologic features and mitotic rate to cytometrically determined DNA content of poorly differentiated lymphocytic lymphomas. Ann NY AcadSci 1986;468:178-194. 28. Richter MN. Generalized reticular cell sarcoma of the lymph nodes associated with lymphocytic leukemia. Am J Pathol 1928,4:285299. 29. Shackney SE. The use offlowcytometry in the diagnosis and biological characterization of non-Hodgkin's lymphomas. Ann NY AcadSci 1986;468:171-177. 30. Shackney SE, Levine AM, Fisher RI, et al. The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases. J Clin Invest 1984;73:1201-1214. 31. Sheibani K, Nathwani BN, Winberg CD, Scott EP, Teplitz RR, Rappaport H. Small lymphocytic lymphoma. Morphologic and immunologic progression. Am J Clin Pathol 1985;84:237-243. 32. Siegelman MH, Cleary ML, Warnke R, Sklar J. Frequent biclonality and Ig gene alterations among B cell lymphomas that show multiple histologic forms. J Exp Med 1985;161:850-863. 33. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-517. 34. Splinter TAW, Noorloos AB, Van Heerde P. CLL and diffuse histiocytic lymphoma in one patient: clonal proliferation of two different B cells. Scand J Haematol 1978;20:29-36. 35. Trump DL, Mann RB, Phelps R, Roberts H, Conley CL. Richter's syndrome: diffuse histiocytic lymphoma in patients with chronic lymphocytic leukemia. A report offivecases and review of the literature. Am J Med 1980;68:539-548. 36. van Dongen JJM, Hooijkaas H, Michiels JJ, et al. Richter's syndrome with different immunoglobulin light chains and different heavy chain gene rearrangements. Blood 1984;64:571-575.
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man's disease: association with Epstein-Barr virus. Am J Pathol 1988;I31:84. 12. Harousseau JL, Flandrin G, Tricot G, Brouet JC, Seligmann M, Bernard J. Malignant lymphoma supervening in chronic lymphocytic leukemia and related disorders. Richter's syndrome: a study of 25 cases. Cancer 1981 ;48:1302-1308. 13. Herman TS, Hammond N, Jones SE, Butler JJ, Byrne GE, McKelvey EM. Involvement of the central nervous system by non-Hodgkin's lymphoma. The Southwest Oncology Group experience. Cancer 1979;43:390-397. 14. JafTe ES. Surgical Pathology of the lymph nodes and related organs. Philadelphia: WB Saunders, 1985. 15. Juneja SK, Cooper IA, Hodgson GS, et al. DNA ploidy patterns and cytokinetics of non-Hodgkin's lymphoma. J Clin Pathol 1986;39:987-992. 16. Lane PK, Townsend RM, Beckstead JH, Corash L. Central nervous system involvement in a patient with chronic lymphocytic leukemia and non-Hodgkin's lymphoma (Richter's syndrome), with concordant cell surface immunoglobulin isotypic and immunophenotypic markers. Am J Clin Pathol 1988;89:254-259. 17. Law IP, Dick FR, Blom J, Bergevin PR. Involvement of the central nervous system in non-Hodgkin's lymphoma. Cancer 1975;36: 225-231. 18. Levitt U, Dawson DM, Rosenthal DS, Moloney WC. CNS involvement in non-Hodgkin's lymphomas. Cancer 1980;45:545-552. 19. Liepman MK, Votaw ML. Meningeal leukemia complicating chronic lymphocytic leukemia. Cancer 1981;47:2482-2484. 20. Long JC, Aisenberg AC. Richter's syndrome. A terminal complication of chronic lymphocytic leukemia with distinct clinicopathologic features. Am J Clin Pathol 1975;63:786-795. 21. Macartney JC, Camplejohn RS, Adler J, Stone MG, Powell G. Prognostic importance of DNA flow cytometry in non-Hodgkin's lymphomas. J Clin Pathol 1986;39:542-546. 22. Mackintosh FR, Colby TV, Podolsky WJ, et al. Central nervous system involvement in non-Hodgkin's lymphoma: an analysis of 105 cases. Cancer 1982;49:586-595. 23. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982.
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