Immunologic and Molecular Biologic Characterization of Pleural Involvement in a Case of T-Chronic Lymphocytic Leukemia

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Dismuke SE, Wagner EH. Pulmonary embolism as a cause of death: the changing mortality in hospitalized patients. JAMA 1986; 255:2039-42 Collins R, Scrimgeour A, Yusuf S, Peto R. Reduction in fatal pulmonary embolism and venous thrombosis by perioperative adm inistration of subcutaneous heparin. N Eng) J Med 1988; 318:1162-73 Dalen JE, Alpert JS. Natural history of pulmonary embolism. Prog Cardiovasc Dis 1975; 17:259-70 Borst RH. Erste Ergebnisse der Notfallbehandlung bei massiver, fulminanter Lungenembolie mit einer rasch injizierten hohen lnitialdosis von Streptokinase. Anaesthesist 1980; 29:39-45 B6ttiger BW, Reim SM, Diezel G. Erfolgreiche Behandlung einer fulminanten Lungenembolie durch hochdosierte Bolusinjektion von Urokinase wahrend der kardiopulmonalen Reanimation. Aniisthesiol Intensivmed Notfallmed Schmerzther 1991; 26:29-36 Kohle W, Pindur G, Stauch M, Rasche H. Hochdosierte Streptokinasetherapie bei fulminanter Lungenarterienembolie [abstract]. Anaesthesist 1984; 33:469 Langdon RW , Swicegood WR, Schwartz DA. Thrombolytic therapy of massive pulmonary embolism during prolonged cardiac arrest using recombinant tissue-type plasminogen activator. Ann Emerg Med 1989; 18:678-80 Scholz KH, Hilmer T, Schuster S, Wojcik J, Kreuzer H, Tebbe U. Thrombolyse bei reanimierten Patienten mit Lungenembolie. Deutsch Med Wschr 1990; 115:930-35 Westhoff-Bieck M, Gulba DC, Claus G, Rafflenbeul W, Lichtlen PR. Lysetherapie bei protrahierter kardio-pulmonaler Reanimation: Nutzen und Komplikationen. [abstract]. Z Kardiol1991; 80(suppl3):139 Neuhof H, Hey D, Glaser E, Wolf H, Lasch HG. Hemodynamic reactions induced by streptokinase therapy in patients with acute myocardial infarction. Eur J Intens Care Med 1975; 1:27-30 Strauer BE. Pathophysiologie und Klinik der Lungenembolie. Internist 1984; 25:108-25 Wester HA, Orellano L, Fenyes-Bellmann J, Hiigl E, Kirchhoff PG. Erfolgreiche Behandlung einer fulminanten Lungenembolie nach 90miniitiger externer Herzmassage. Deutsch Med Wschr 1986; 111:1151-54 Cere) A, Burger AJ. The diagnosis of a pulmonary artery thrombus by transesophageal echocardiography. Chest 1993; 103:944-45 Horstkotte D, Heintzen MP, Strauer BE. Kombinierte mechanische und thrombolytische Wiederer6ffnung der Lungenstrombahn bei massiver Lungenarterienembolie mit kardiogenem Schock. Intensivmed 1990; 27:124-32 Eisenmann B, WeissE, Greff D, Desroche P, Bauer MC, Kretz JG, et al. Massive Lungenembolie: Operationsindikationen und Ergebnisse. Internist 1984; 25:126-29 Gray HH, Morgan JM, Paneth M, Miller GAH. Pulmonary embolectomy for acute massive pulmonary embolism: an analysis of 71 cases. Br Heart J 1988; 60:196-200 Gramann J, Lange-Braun P, Bodemann T, Hochrein H. Einsatzm6glichkeiten der Thrombolyse in der Reanimation. Intensivmed 1988; 25:425-29 Hopf HB, Flo,Bdorf T, Breulmann M. Rekombinanter Gewebeplasminogenaktivator (rt-PA) zur Notfallbehandlung der perioperativen lebensbedrohlichen Lungenembolie (Stadium IV). Anaesthesist 1991; 40:309-14 MolinaJE, Hunter DW, YedlickaJW, Cerra FB. Thrombolytic

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Immunologic and Molecular Biologic Characterization of Pleural Involvement in a Case of T -Chronic Lymphocytic Leukemia* Ferenc Szalay, M.D.; Miklos Szathmari, M.D.; Katalin Pillbczi, M.D.; janos Foldi, Ph.D.; and judith Demeter, M.D.

Pleural involvement is a rare complication of chronic lymphocytic leukemia (CLL). We report a CLL case of T-cell origin (documented by cell surface marker as well *From the First Defartment of Medicine, Semmelweis University Medical Schoo (Drs. Szalay, Szathmari, and Demeter) and National Institute of Haematology and Blood Transfusion (Drs. Pal6czi and Foldi), Budapest, Hungary. CHEST / 106 / 4 / OCTOBER, 1994

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as DNA rearrangement studies) where the lymphoid cells of the pleural fluid were found to belong to the same monoclonal population of T cells as those of the (Chest 1994; 106:1283-85) peripheral blood. CLL=chronic lymphocytic leukemia; Ig=immunoglobulin; MoAb=monoclonal antibody

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leural involvement is not a characteristic complication of chronic lymphocytic leukemia (CLL), although pleural fluid has been found in 40 percent of autopsy cases. 1 Chronic lymphocytic leukemia is usually the neoplastic disorder of B lymphocytes. Lymphoid neoplasms ofT-cell origin account for less than 20 percent of human lymphoproliferative malignancies. Within the spectrum of peripheral T-celllymphomas, true T-cell CLL occurs very rarely (3 of 75 cases in a recent series).2 Pulmonary and pleural involvement was diagnosed in vivo in 5 of these 75 peripheral T-celllymphoma cases. 2 As far as we know, this is the first report on immunologically and molecular biologically documented pleural involvement in T-cell CLL. We describe a patient with T-cell CLL who was shown to have pleural involvement consisting of the same neoplastic clone as the underlying lymphoma.

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CASE REPORT

A 48-year-old man was known to suffer from CLL since Ma y 1990. The diagnosis was based on the hepatosplenomegaly, generalized lymphadenopathy, marked lymphocytosis with bone marrow infiltration, and absence of a mediastinal mass. The neoplastic cells were medium sized, with a thin rim of agranular cytoplasm . An enzyme-linked immunosorbent assay test for human T-celllymphotropic virus type 1 antibodies was negative. Because of progressive, Rai stage IV disease, he was treated with cytostatic drugs according to the CVP (cyclophosphamide, vincristine, prednisolone) protocol. Further progression and skin infiltration developed prompting an immunologic evaluation in October 1991. The patient was found to suffer from T-cell CLL as described below. At the end of August 1991, recombinant interferon alfa-2b (Intron-A, Schering-Plough, Memphis, Tenn) therapy was started at a dose of 3 million U per day for a week, followed by 3X3 million U of recombinant interferon alfa-2b weekly. Histologically proven cutaneous infiltrates of the dermis promptly regressed. Two months later, during treatment with recombinant interferon alfa-2b, the patient was found to have developed a large left-sided pleural effusion. On puncture it proved to be bacteriologically sterile exudate. The pleural exudate had a leukocyte count of 60 g/ L. Virtually all these cells were lymphocytes, made up by mature T cells. The following treatment modalities have been tried unsuccessfully: interferon alfa-2b subcutaneously as a single agent and combined with cytostatic drugs , repeated cycles of combined cytostatic treatment containing prednisone, methotrexate, leucovorin, doxorubicin (Adriamycin), cyclophosphamide, etoposide, cytarabine, bleomycin, and vincristine (Pro-MACE Cyta-BOM), intrapleural methotrexate, and lastly splenic irradiation. Intrapleural methotrexate was found to prevent recurrence of the pleural effusion for 10 weeks. Terminally massive bilateral pleural effusion and ascites developed, repeated intrapleural and intraperitoneal methotrexate therapy was unsuccessful, and the patient died in March 1992, 5 months after the first appearance 1284

4_3 FIGURE l. Autoradiograph of Southern blot of DNA samples digested with EcoRI and hybridized with C ~-probe (Hind III and Bam HI enzymes were not informative). N=normal control; PL and PB=pleural fluid and peripheral blood of the index patient, respectively. The numbers indicate the size of fragments in kilobase pairs.

of the pleural effusion. On autopsy bilateral pleural effusions, emphysema, pleuritis fibrinosa, ascites, and generalized lymphadenopathy were found with diffuse lymphoid infiltrates in the bone marrow; the spleen weighed 2,280 g; the histology was consistent with the diagnosis of CLL. MATERIALS AND METHODS

Isolation of Mononuclear Cells From the Peripheral Blood and the Pleural Effusion and Immunophenotyping Mononuclear cells were isolated from the heparinized peripheral blood and the pleural eff usion was isolated on a FicolUromiro gradient. Interphase cells were washed twice and resuspended in RPMI 1640 containing 10 percent fetal calf serum (Gibco, United Kingdom) and antibiotics. The T-cell-associated monoclonal antibodies (MoAbs) used in this study were the following: T6 (CD1), Tl1 (CD2), T3 (CD3), T4 (CD4), T8 (CD8) (Ortho Diagnostic Systems), and T1 (CD5, Leu- I) (Becton-Dickinson). The B-cell-associated reagents used were B4 (CD19) (Coulter) and slgM. Class II antigens were detected with an HLA-DR reagent (Becton-Dickinson). Mo-2 (CD14) (Coulter) is a marker of monocytes, while CD25 is an activation marker with Pleural Involvement in T -Chronic Lymphocytic Leukemia (Szalay eta/)

anti-IL-2 receptor activity (Ortho Diagnostic Systems). Binding of various MoAbs was assessed with indirect immunofluorescence using FITC-conjugated rabbit antimouse immunoglobulin (DAKO) as second antibody. Cells prepared the same way, but without the addition of the first layer antibody, served as negative controls. The anti-IgM antibody (Heintel) was used as directly conjugated with FITC. Analysis of 10,000 lymphoid cells was done using a How cytometer (F ACStar, Becton-Dickinson).

DNA Extraction and Southern Blotting DNA was extracted from the peripheral blood mononuclear cells as described previously.s· 4 The configuration of the T-cell receptor chain genes was investigated by probing EcoRI and Hind III DNA digests with a C B-probe M131B10BBl. 3 The configuration of the immunoglobulin (Ig) heavy chain locus was analyzed by the Ig heavy chain probe M12C76R51A after digestion of DNA with EcoRI and Hind III restriction endonucleases. All DNA probes originated from the same source (T.H. Rabbits, Cambridge, United Kingdom ). RESULTS

The patient was found to suffer from T-cell CLL (CD4+, 98 percent) both by cell surface marker as well as DNA rearrangement studies. Analysis of genomic DNA of peripheral blood mononuclear cells showed the presence of a monoclonal population of T cells, the T -cell receptor /3-chain gene being rearranged on both alleles while the Ig heavy chain genes were in germline configuration. Cell surface marker and DNA studies on the mononuclear cells isolated from the pleural fluid showed this population of lymphoid cells to carry the same cell surface marker and DNA rearrangement characteristics as the cells of the peripheral blood. They were mature T cells (CD2+, 69 percent; CD3+, 78 percent; CD4+, 79 percent; CD8, 16.9 percent; CD5, 34.9 percent), lacked the immature T -cell marker CDl , while B cells represented just a minority of the cell population (CD19 °, 0.5 percent; slgM, 8.4 percent). Results of DNA studies showing a monoclonal population of T cells both in the pleural fluid and in the peripheral blood are shown in Figure l. DISCUSSION

Patients with malignant lymphoma not infrequently develop benign lymphoid-rich effusions. To help the differential diagnosis of malignant from benign pleural effusions, acomputerized interactive morphometry system has been developed.5 While the predictive value of this expert system exceeded 90 percent in the diagnosis of effusions accompanying malignant lymphoma or benign pleural lymphocytosis, nevertheless the system was unsuitable for the diagnosis of malignancy in effusions from patients with CLL. 5 Lymphoproliferative disease was ascertained by molecular analysis of the mononuclear cells of the pleural effusion in a case of non-Hodgkin's lymphoma without palpable lymph nodes.6 The results of our DNA studies performed simultaneously on the mononuclear cells from the peripheral blood and from the pleural fluid led us to the conclusion that the neoplastic cells in the pleural fluid had the same clonal origin as the leukemic process itself. In conclusion, cell surface marker and DNA studies of the pleural fluid mononuclear cells appear to be appropri-

ate for the definition of the cellular origin of the effusion in patients with CLL. ACKNOWLEDGMENT: This work has been supported by a grant of the Ministry of Health and Welfare, Hungary ETT 12M-008/ 1990. REFERENCES

1 Klatte CE, Yardley J, Smith EB, Rohn R, Campbell JA. The pulmonary manifestations and complications of leukemia. AJR 1963; 89:598-609 2 Chott A, Augustin I, Wrba F, Han ak H, Ohlinger W, Radaskiewicz T. Peripheral T-celllymphomas: a clinicopathological study of 75 cases. Hum Pathol1990; 21:1117-25 3 Foroni L, Foldi J, Matutes E, Catovsky D, O'Connor NJ, Baer R, et al. a , (3 and 'Y T-cell receptor genes: rearrangements correlate with haematological phenotype in T cellleukaemias. Br J Haematol1987; 67:307-18 4 Demeter J, Pal6czi K, Foldi J, Hokland M, Hokland P, Benczur M, etal. Immunological and molecular biological identification of a true case ofT-hairy cell leukaemia. Eur J Haematol1990; 43:339-45 5 Walts A, Marchevsky AM. Computerized interactive morphometry: an expert system for the diagnosis of lymphoid-rich effusions. Am J Clin Patholl989; 92:765-72 6 Cavallero GB, Bonferroni M, di Celie PF, Gallamini A, Foa R. Pleural effusion in a case of non-Hodgkin 's lymphoma: diagnostic use of molecular analysis. Recent Prog Med 1990; 81:568-70

Atrial-Esophageal Fistula Shown by Transthoracic Echocardiogram* Paiboon Mahaisavariya, M.D.; Richard M. Deits, M.D.; Thomas P. Cowell, M.D. ; and Shelley M. Shapiro, M.D ., Ph.D.

Nontraumatic atrial-esophageal fistula is a catastrophic problem usually diagnosed postmortem and almost invariably fatal. We report the first case of a patient in whom the diagnosis of atrial-esophageal fistula was made from a transthoracic echocardiography antemortem. Echocardiography showed multiple microbubbles in the left atrium and ventricle emanating from the posterior aspect of the left atrium adjacent to the pulmonary veins. The literature is reviewed and the significance of the case and the echocardiogram is discussed. (Chest 1994; 106:1285-88} GI =gastrointestinal

Key words: esophageal ulcer, gastroesophageal-atrial fistula *From the Division of CardiologY-, Harbor-UCLA Medical Center UCLA School of Medicine (Drs. Mahaisavariya, Deits, and Shapiro), Department of Cardiology (Dr. Deits) and Department of Pathology (Dr. Cowell), Torrance Memorial Hospital; Torrance, Calif. Funded in part by the Saint Johns Cardiovascular Research Center. CHEST /106/4/ OCTOBER, 1994

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