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Persistent Lymphocytosis with Chromosomal Evidence of Malignancy
CASE
REPORTS
Persistent Lymphocytosis with Chromosomal Evidence of Malignancy
JEROMEI. BRODY, M.D. RICHARDA. BURNINQHAM,M.D. PETERC. NOWELL,M.D. DAVID T. ROWLANDS,Jr., M.D. PAUL FREIBURG,M.D. RONALD P. DANIELE,M.D. PhkxMph~,
Pennsylvsnia
From the Department of Medlclne, The Qraduate Hoapltal of the University of Pennsylvania, the Departments of Medlclne and Pathology, the Unlverslty of Pennsylvanla School of Medlclne, and the Medlcal Servlce, U.S. Naval Hospital, Phlladelphla, Pennsylvania. Thls study was eupported by U.S. Public Health Service Research Grants CA-07000 (J.I.B.), CA-12778 (P.C.N., D.T.R. and R.P.D.), AM-14372 (P.C.N., D.T.R. and R.P.D.), U.S. Public Health Research Career Development Award Ca 8371 (J.I.B.), and by the Bureau of Medicine and Surgery, U.S. Navy CICC Project No. 3-05-123. Requests for reprlnts should be addressed to Dr. Jerome I. Brody, Department of MedIcIns, Qraduate Hos-
pltal of the Unlversltyof Pennaykanla,Phlladelphla, PennsylvanIa19148.Manuscrlptaccepted April 5, 1974.
An Inappropriate, sustained and absolute lymphocytosls In a young man was Investigated uslng technics which attempted to define the more detalled features of these cells and their 1 and B subpopulatlons. The results of the tests for lymphocyte RFC (E) and complement (EAC) rosette formatlon, Immunofluorescence, ln vitro phytohemagglutlnln (PHA) stlmulatlon and 14C-cyclophosphamlde blndlng assays, when evaluated In a combined and Interrelated fashion, lndlcated that the lymphocytosls was malnly but not solely, due to an absolute elevation In circulating 1 lymphocytes. Moreover, an aneuplold cell line wlth 47 chromosomes, presumably T cells, also was detected In PHA-stlmulated cultures. These data, associated wlth the patlent’s anemla, lymphocyte-lnflltrated bone marrow, hepatosplenomegaly, pharyngeal lymphold tumor and the clonal prollferatlon of lymphocytes bearIng an abnormal karyotype, furnish evidence that the lymphocytosls Is more than an unremlttlng leukemold reactlon and Is potentlally neoplastlc. The detectlon of the chromosomal abnormallty at thls patlent’s age and polnt In disease may make hlm especlally unlque In that he could be a cllnlcal prototype for other patlents wlth lymphoretlcular tumors which remain undetected at their onset. We describe a young man with a sustained, absolute lymphocytosis, detected fortuitously, and occurring at an age and under circumstances which make this type of ieukocytosls singularly inappropriate. Failure to associate this abnormality with the more common, self-limited causes of iymphocytosis, as the ciinlcai history will indicate, and the inabllity of routine laboratory examinations to provide an acceptable explanation for the increase in peripheral blood lymphocytes, provoked further investigation with technics which define the more detailed features of these ceils and their T and B subpopulations [ 11. Data provided by these methods supported the diagnosis of a lymphoproilferative disorder in this patient best characterized as a potentially malignant iymphocytosis in which the predominant, but not necessarily the sole, participant is the T ceil.
CASE REPORT A 21 year old white man was admitted to the United States Naval Hospital, Philadelphia, Pa., In September 1970 because of a cellulltls that developed after a puncture wound of the foot. Aside from signs of infection, which responded promptly to antlblotics, the findlngs on physlcal examina-
April 1975 The American Journal of Medlclne Volume58
547
LYMPHOCYTOSIS
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
ET AL
Figure 1. Appearance of the large, atypical lymphocytes in the peripheral blood. Original magnification X 1,000, reduced by 38 per cent.
Figure 2. Lymphocytes and plasma cells in the bone marrow aspirate. Original magnification X 1,000, reduced by 38 per cent.
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tion were unremarkable, and lymphadenopathy and hepatosplenomegaly were absent. Similarly, there was nothing noteworthy in the patient’s past, personal or family histories except his statement that minor skin abrasions, sores and cuts frequently would become infected. The results of the initial hemogram were surprising and unanticipated, showing a hemoglobin level of 13 g/100 ml, hematocrit value 39 per cent, platelet count 282,000/mm3, reticulocyte count 1.3 per cent and a total white blood cell count of i6,000/mm3, of which 99 per cent were lymphocytes (Figure 1). Additional normal laboratory studies included fasting blood sugar, blood urea nitrogen, serum electrolytes, calcium, uric acid, creatinine, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase (LDH), alkaline phosphatase, serum bilirubin and urinalysis. The albumin was normal by serum protein electrophoresis, but diffuse hypergammaglobulinemia was present. Immunoelectrophoresis did not show a paraprotein, and the immunoglobulins, quantitated by radial immunodiffusion (Immunoplates, Hyland Laboratories Division of Travenol, Costa Mesa, Calif.) were IgG 2,160 mg/lOO ml (normal 1,120 f 250 mg/lOO ml), IgA 660 mg/lOO ml (normal 280 f 60 mg/ 100 ml) and IgM 190 mg/ 100 ml (normal 120 f 30 mg/ 100 ml). A serologic test for syphilis (VDRL), fluorescent antinuclear antibody test, Coombs’ test, heterophil antibody, tests for cryoglobulins and cryofibrinogen were all negative. The chest x-ray film was normal, although a liver-spleen scan performed with ggmT~ sulfur colloid showed both organs to be enlarged. The bone marrow could not be aspirated, but a biopsy specimen was diffusely infiltrated with lymphoid cells. The skin test with mumps antigen was positive, although there was no response to intermediate strength purified protein derivative, histoplasmosis, coccidioidomycosis and blastomycosis antigens. Sensitization to dinitrochlorobenzene (DNCB) was not performed. Because of the patient’s rapid and uneventful recovery, he was discharged to be observed as an outpatient. During the ensuing 18 months his hemoglobin level varied from 11.8 to 14.0 g/100 ml and his total white blood cell count fluctuated between 11,000 and 18,500/mm3, with 90 to 99 per cent lymphocytes. In April 1972, he again entered the hospital complaining of a sore throat and hemoptysis. A large, ulcerated, polypoid lesion was seen filling the left valecula which, by biopsy, was shown to consist of lymphocytes and plasma cells. Two weeks later the mass resolved spontaneously and could not be seen on repeat laryngoscopy. No pathogenic organisms grew in nose and throat cultures. Serum protein electrophoresis, immunoelectrophoresis and quantitative immunoglobulins were unchanged. The patient left the hospital to be kept under medical surveillance. In February 1973, after a year during which he had six different episodes of sore throats, one caused by P-hemolytic Streptococcus rapidly responding to penicillin, he was admitted to the hospital for reevaluation. In addition to repetition of the earlier studies, the results of which actually remained stable, a bone survey and a scan performed with ggmTc polyphosphate were reported as unremarkable. The bone marrow could be aspirated at this time
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
ET AL.
and, in various areas, contained a mixture of lymphocytes and plasma cells (Figure 2). A paucity of immature and mature granulocytic precursors was seen, but erythroid and megakaryocyte developmental forms were adequate in number. The immunoglobulin levels again were elevated, a paraprotein was not found and isohemagglutinins were normal. The patient was referred to the Division of Hematology of the Graduate Hospital of the University of Pennsylvania and the Department of Pathology of the University of Pennsylvania School of Medicine for more detailed investigation of his circulating lymphocytes. METHODS AND MATERIALS In all the studies to be described lymphocytes were separated by differential and Ficoll-Hypaque density gradient centrifugation [2,3] when the patient’s total white cell count was 18,500/mm3, with 97 per cent lymphocytes. Formation of lymphocyte rosettes was studied using previously detailed methods [4,5]. Phagocytic cells were removed from heparinized, whole peripheral blood by a 30 minute incubation with carbonyl iron after which the blood was sedimented in 5 per cent dextran [6]. The lymphocytes then were separated as indicated earlier. Erythrocyte (RFC, E) rosettes were formed when equal volumes of washed sheep red blood cells (0.5 per cent) were mixed with lymphocytes (4 X lo6 cells/ml) in Hanks Balanced Salt Solution (HBSS) in microtiter plates (Cooke Engineering, Alexandria, Va.). These preparations then were centrifuged at 200 g for 5 minutes at 22’C, incubated for an additional hour at 22’C, gently resuspended and studied microscopically in a hemocytometer. A rosette was defined by adherence of 3 or more sheep red blood cells to a lymphocyte. EAC (complement) rosettes were prepared using sheep red blood cells incubated at 37’C for 30 minutes with 19s rabbit antibody to Forsmann antigen diluted 1: 1000 with HBSS. These sensitized cells then were kept at 37OC for 30 minutes with a 1:lO dilution of serum from normal C3H mice as a source of complement. Finally, these EAC cells (0.5 per cent suspension) were mixed in equal volumes with lymphocytes (0.1 ml), centrifuged at 200 g at 37’C, resuspended and evaluated as just outlined. To quantitate lymphocyte immunofluorescence, the cells were prepared as for rosettes and suspended in HBSS at 10 X 1O6 cells/ml. Then 0. I ml aliquots were incubated for 30 minutes at 37OC with 0.1 ml of monospecific rabbit antihuman serum immunoglobulin (G,A,M,D,E,k) components obtained commercially (Meloy, Falls Church, Va.). These preparations were washed 3 times and mounted in a 9:l glycerol:buffered saline solution medium. The slides were examined immediately using a Leitz fluorescent microscope with a Ploem attachment and results recorded as per cent positive cells. To evaluate mitogen response, cultures contained lo6 lymphocytes in 1 ml Modified Eagles Medium with 10 per cent human AB serum and 50 pg/ml phytohemagglutininM (PHA) (Difco, Detroit, Mich.). Proliferation was measured at 3, 5 and 7 days in duplicate cultures by the addition of tritiated thymidine (0.25 &i) 16 hours before termination of the culture and counting by standard scintillation methods.
April 1975
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LYMPliOCYtOSlS
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
Chromosome preparations were made from 3 and 7 day cultures. Colchicine was added 3 hours before termination, and the air-dried chromosome preparations were stained with aceticorcein [7]. Finally, a recently described assay which measures the binding of 14C-alkylating agents to lymphocytes [8] also was performed as a further means of distinguishing between T and B cells. Previous and ongoing experiments in this laboratory have demonstrated that the B cells in conventional chronic lymphocytic leukemia bind less 14C-labelled alkylating compounds than their T cell counterparts [8,9]. On this basis, 12 pg 14C-cyclophosphamide (14CCTX) (speclflc actlvlty 7.6 mCl/mmol, New England Nuclear, Boston, Mass.), a concentration shown to be cytotoxlc in vitro [lo], and approximating the cllnically employed dosage [ 111, were added for 2 hours to 48 hour old, unstimulated lymphocyte cultures In TC 199 (Dlfco, Detroit, Mlch.) and 30 per cent autologous plasma. The cells were washed twice with saline solution, the entlre pellet was digested In Hyamlne (Rohm & Haas, Philadelphia, Pa.) and then transferred to vlals for counting by sclntlllation spectrometry. Mitogen stlmulatlon and 14C-CTX bindlng also was accompllshed with lymphocytes from the patient’s parents and two sisters none of whom had physical or laboratory abnormalities. RESULTS The results which follow must be evaluated in terms of absolute numbers and relative proportions of lymphocyte subgroups, both in normal as well as in the patient’s blood. It should be acknowledged that multiple surface marker assays vary within different laboratories and are not always in agreement, and that the cells under study do not necessarily retain every feature ascribed to their normal counterparts. These are the reasons additional methods of differentlation are being devised, as is true In this laboratory with the Cytoxan measurement, and as holds for the preparation of antithymocyte antiserums which, up to the present, has enjoyed limited use and availability. There were 38 per cent peripheral lymphocytes which formed RFC (E) rosettes (normal 42 to 66 per cent). With approximately 18,000 patlent lymphocytes/mm3 this represents a total of 6,800 RFC/mm3 (rounded figures), or greater than three times the maximum number expected in normal blood (2,200/ mm3), based on a total normal leukocyte count of 10,000/mm3 and 33 per cent lymphocytes. Thus, the patient has an absolute T cell lymphocytosls when compared with the normal value. However, with the RFC method, his T cells are slightly below the minimum absolute number of 7,500/mm3 (rounded figures) anticipated In his own blood. Only 1 per cent of his lymphocytes developed EAC rosettes (normal 8 to 17 per cent), with an absolute value of 180/mm3. This is just within the range of 100 to 560/mm3 for normal blood, but far below the 1,430 to 3,050/mm3 expected with this patient’s
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ET AL.
total lymphocyte count. Therefore, with this technic at least, his B cells are not increased. The immunofluorescence measurements showed staining with IgG 4 per cent (normal 6 to 10 per cent), IgA 2 per cent (normal 4 to 10 per cent), IgM 6 per cent (normal 6 to 14 per cent), IgD 3 per cent (normal 1 to 10 per cent), IgE 0 per cent (normal 2 to 5 per cent) and k 0 per cent (normal 7 to 24 per cent). There were 2,700/mm3 lymphocytes (rounded figures) carrying immunoglobulin receptors, somewhat above the maximum of 1,600/mm3 allowed in normal blood, but below the minimum of 3,400/mm3 for the patient’s own circulation. Since some of the positive cells may reflect adsorbed immunoglobulins from the serum [ 121, the immunofluorescence values may be spuriously high. This outcome may now be avoided by exposing the lymphocytes to trypsin, which removes the extraneous surface protein [ 131, but this was not being used at the time of this study. Nevertheless, this leaves 7,200/mm3 lymphocytes unaccounted for. The PHA cultures, a usually accepted test of T cell presence and reactivity, demonstrated a proliferation curve comparable to that seen in normal subjects: 41,000 dpm on day 3, followed by a decrease to 6,000 on day 5 and 1,300 on day 7. In contrast, lymphocytes from patients with chronic lymphocytic leukemia, now considered a B lymphocytosis, consistently show a delayed and reduced PHA response. Adequate chromosome preparations were obtained from PHA-stimulated cultures. Chromosome counts were made on a total of 100 cells: 67 from 3 day PHA cultures and 33 from 7 day PHA cultures. The majority were diploid (79 cells, 46 chromosomes; 2 cells, less than 46 chromosomes), but an aneuploid subpopulation (19 cells) with 47 chromosomes was present in both the 3 and 7 day cultures. The aneuploid cells were generally of poor technical quality, a common finding with neoplastic cells In chromosome preparations [ 141, but karyotype analysis revealed the extra chromosome to be a member of group C (chromosome 6-12 and the X, Figure 3). No other significant abnormality was noted in karyotype analyses of cells with either 46 or 47 chromosomes, the large size of the Y chromosome being considered a normal variant. The binding of 14C-CTX by lymphocytes from 13 normal donors gave a mean of 324 cpm (range 172 to 623 cpm). The normal 14C-CTX radioactivity on the patient’s lymphocytes (435 cpm) is consistent with their identification as that of a T lymphocyte population. In contrast, 14C-CTX binding of lymphocytes from 11 patients with conventional chronic lymphocytic leukemia [ 15,161 gave a mean of 76 cpm (range 17 to 109 cpm). Lymphocytes from parents and siblings were normal. Additional evidence
LYMPHOCYTOSIS
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
ET AL.
[9], not detailed here, supporting the T cell’s affinity for Cytoxan, comes from membrane alteration with neuraminidase, an enzyme which removes terminal sialic acid from the cell membrane by cleaving 2,3, and 2,6 ketoglycosidic linkages of the cell surface mucopolysaccharides [ 171. This maneuver markedly decreased “C-CTX binding to normal lymphocytes and artificially converted them, in a qualified way, to a form simulating B cells, which inherently contain less membrane sialic acid than their T cell relatives [18]. It is not inappropriate to assume, therefore, that those cells previously escaping recognition have expressed their presence through mitogen response and alkylating agent binding. COMMENTS Although this patient’s clinical behavior generally has not been consistent with an overt neoplasm, the lymphocyte-infiltrated bone marrow, intermittent anemia, hepatosplenomegaly and pharyngeal lymphoid tumor furnish evidence that his lymphocytosis is more than merely a benign unremitting leukemoid reaction. Furthermore, the apparent clonal’ proliferation of lymphocytes being an abnormal karyotype strongly suggests that this is a potentially malignant condition. It is difficult to be unequivocally certain as to which lymphocyte subgroup is primarily responsible for the raised total lymphocyte count. Taken together, the various procedures described indicate an absolute increase in circulating T cells, and also a proportion of null cells which could not be characterized as T or B features with the technics used here. With newer methods this outcome, especially with normal lymphocytes, will become less frequent. The functional capacities of both the T and B cell appear to be retained in vivo, at least in part. The response to mumps antigen is evidence of T cell competence, although this may not hold for all antigens. Whereas the hyperimmunogammaglobulinemia is reasonable evidence that the plasma cells in the marrow and elsewhere are secretory, the specificity and avidity of these proteins for infectious agents have not been defined. The cytogenetic data suggest that at least a proportion of the lymphocytes responding to PHA in culture (presumably T cells) are neoplastic, as indicated by the aneuploid cell line. The poor technical quality of the aneuploid metaphases, as compared with diploid cells on the same slides, argues for their neoplastic character [ 141 and probably also resulted in an underestimation of their frequency. It is possible, _ The term clonal is used in the cytogenetic sense and refers to a population of ceils with the same karyotype, presumably descendant from a single precursor. l
Figure from 3 of 100 somes,
3. Karyotype wtih extra c’ group chromosome day PHA-stimulated lymphocyte culture. Nineteen metaphases from PHA cultures had 47 chromosuggesting a neoplastic clone of T ceils.
of course, that this 47, XY, C+ clone reflects constitutional mosaicism (e.g., an XXY subpopulation) rather than a neoplastic process, but the absence of any stigmata of Klinefelter’s syndrome and the different quality of these cells from the diploid population make this conclusion seem very unlikely. If adequate preparations can be obtained from this patient in the future, autoradiographic and banding methods will permit precise identification of the extra C group chromosome and should definitely resolve this question. The lifelong history of infection provides a speculative explanation for the events described and the laboratory results obtained. It is conceivable that, initially, a general defect existed in leukopoiesis, such that abnormal differentiation of the totipotent stem cell led to persistent granulocytopenia associated with or followed by a distorted mechanism regulating the number of circulating lymphocytes [ 191. Detection of the chromosomal aberration at this patient’s present age and point in disease may make this patient especially unique in the sense that he could be a clinical prototype for patients with other lymphoreticular tumors which also may begin in a clonal fashion but are not always recognized as such because of a more subtle cellular change not measurable by present methods. Whether, as is applicaApril 1975 The AmericanJournalof Medicine Volume58
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LYMPHOCYTOSIS WITH CHROMOSOMAL EVIDENCE OF MALIGNANCY-BRODY
ble in this instance, too, these clones consistently become overtly destructive may depend on the integrity of host factors such as immunosurveillance, and on any further alterations which may occur in the abnormal cell group.
ET AL.
ACKNOWLEDGMENT Grateful thanks are extended to Mrs. Jane Kohut, Mrs. Mary Haidar and the Misses Julie Jensen and Paula Henry for their technical assistance.
REFERENCES
4.
5. 6.
7.
8.
9. 10.
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Aisenberg AC, Bloch KJ: lmmunoglobulins on the surface of neoplastic lymphocytes. N Engl J Med 287: 272, 1972. Brody JI, Beizer LH: Alteration. of blood group antigens in leukemic lymphocytes. J Clin Invest 44: 1582, 1965. Bdyum A: Separation of leukocytes from blood and bone marrow: with special reference to factors which influence and modify sedimentation properties of hematopoietic cells. Stand J Clin Lab Invest 21 (suppl 97): 1, 1968. Medes NF, Tolnai MEA, Silveria NPA, Gilbertson RB, Metzgar RS: Detection and specific depletion of subpopulations of human lymphocytes by rosette formation with antibody-complement sensitized erythrocytes and unsensitized erythrocytes. J lmmunol 111: 860, 1973. Daniele RP, Rowlands DT Jr: In preparation. van Rood JJ, van Leeuwen A, Zweerus R: 4a and 4b antigen, do they or don’t they? Histocompatibility Testing (Terasaki P, ed), Copenhagen, Munksgaard. 1970, p 93. Moorehead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA: Chromosome preparations of leucocytes cultured from human peripheral blood. Exp Cell Res 20: 613, 1960. Brody JI: Diminished binding of alkylating agents by lymphocytes in chronic lymphocytic leukemia. J Clin Invest 52: 13a, 1973. Brody JI: Differential uptake of alkylating agents by normal and leukemic lymphocytes. Cancer (in press). Singer BL, Brody JI: In vitro activation of cyclophosphamide
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11.
12.
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17.
18.
19.
by phytohemagglutinin-stimulated lymphocytes. Am J Med Sci 264: 307,1972. De Vita VT, Serpick AA, Carbone PP: Combination chemotherapy in the treatment of advanced Hodgkin’s disease. Ann Intern Med 73: 881, 1970. Preud’homme JL, Seligman M: Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40: 777, 1972. Rowlands DT Jr, Danielle RP, Nowell PC, Wurzel HA: Characterization of lymphocytic subpopulations in chronic lymphocytic leukemia. Cancer (in press). Nowell PC, Hungerford DA: Chromosome changes in human leukemia and a tentative assessment of their significance. Ann NY Acad Sci 113: 654, 1964. Brody JI, Oski FA, Singer DE: Impaired pentose phosphate shunt and decreased glycolytic activity in lymphocytes of chronic lymphocytic leukemia. Metabolic pathway? Blood 34: 421, 1969. Brody JI: Augmentation of leukemic lymphocyte reactivity to phytohemagglutinin by salivary receptor. Blood 40: 70, 1973. Grothaus EA, Flye MW, Yunis E, Amos DB: Human lymphocyte reactivity modified by neuraminidase. Science 173: 542,197l. McClelland DA, Bridges JM: The total n-acetyl neuraminic acid content of human normal and lymphocytic leukemic lymphocytes. Br J Cant 27: 114. 1972. Vincent PC, Gunz FW: Control of lymphocyte level in the blood. Lancet 2: 342, 1970.
REPORTS
Persistent Lymphocytosis with Chromosomal Evidence of Malignancy
JEROMEI. BRODY, M.D. RICHARDA. BURNINQHAM,M.D. PETERC. NOWELL,M.D. DAVID T. ROWLANDS,Jr., M.D. PAUL FREIBURG,M.D. RONALD P. DANIELE,M.D. PhkxMph~,
Pennsylvsnia
From the Department of Medlclne, The Qraduate Hoapltal of the University of Pennsylvania, the Departments of Medlclne and Pathology, the Unlverslty of Pennsylvanla School of Medlclne, and the Medlcal Servlce, U.S. Naval Hospital, Phlladelphla, Pennsylvania. Thls study was eupported by U.S. Public Health Service Research Grants CA-07000 (J.I.B.), CA-12778 (P.C.N., D.T.R. and R.P.D.), AM-14372 (P.C.N., D.T.R. and R.P.D.), U.S. Public Health Research Career Development Award Ca 8371 (J.I.B.), and by the Bureau of Medicine and Surgery, U.S. Navy CICC Project No. 3-05-123. Requests for reprlnts should be addressed to Dr. Jerome I. Brody, Department of MedIcIns, Qraduate Hos-
pltal of the Unlversltyof Pennaykanla,Phlladelphla, PennsylvanIa19148.Manuscrlptaccepted April 5, 1974.
An Inappropriate, sustained and absolute lymphocytosls In a young man was Investigated uslng technics which attempted to define the more detalled features of these cells and their 1 and B subpopulatlons. The results of the tests for lymphocyte RFC (E) and complement (EAC) rosette formatlon, Immunofluorescence, ln vitro phytohemagglutlnln (PHA) stlmulatlon and 14C-cyclophosphamlde blndlng assays, when evaluated In a combined and Interrelated fashion, lndlcated that the lymphocytosls was malnly but not solely, due to an absolute elevation In circulating 1 lymphocytes. Moreover, an aneuplold cell line wlth 47 chromosomes, presumably T cells, also was detected In PHA-stlmulated cultures. These data, associated wlth the patlent’s anemla, lymphocyte-lnflltrated bone marrow, hepatosplenomegaly, pharyngeal lymphold tumor and the clonal prollferatlon of lymphocytes bearIng an abnormal karyotype, furnish evidence that the lymphocytosls Is more than an unremlttlng leukemold reactlon and Is potentlally neoplastlc. The detectlon of the chromosomal abnormallty at thls patlent’s age and polnt In disease may make hlm especlally unlque In that he could be a cllnlcal prototype for other patlents wlth lymphoretlcular tumors which remain undetected at their onset. We describe a young man with a sustained, absolute lymphocytosis, detected fortuitously, and occurring at an age and under circumstances which make this type of ieukocytosls singularly inappropriate. Failure to associate this abnormality with the more common, self-limited causes of iymphocytosis, as the ciinlcai history will indicate, and the inabllity of routine laboratory examinations to provide an acceptable explanation for the increase in peripheral blood lymphocytes, provoked further investigation with technics which define the more detailed features of these ceils and their T and B subpopulations [ 11. Data provided by these methods supported the diagnosis of a lymphoproilferative disorder in this patient best characterized as a potentially malignant iymphocytosis in which the predominant, but not necessarily the sole, participant is the T ceil.
CASE REPORT A 21 year old white man was admitted to the United States Naval Hospital, Philadelphia, Pa., In September 1970 because of a cellulltls that developed after a puncture wound of the foot. Aside from signs of infection, which responded promptly to antlblotics, the findlngs on physlcal examina-
April 1975 The American Journal of Medlclne Volume58
547
LYMPHOCYTOSIS
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
ET AL
Figure 1. Appearance of the large, atypical lymphocytes in the peripheral blood. Original magnification X 1,000, reduced by 38 per cent.
Figure 2. Lymphocytes and plasma cells in the bone marrow aspirate. Original magnification X 1,000, reduced by 38 per cent.
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April 1975
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tion were unremarkable, and lymphadenopathy and hepatosplenomegaly were absent. Similarly, there was nothing noteworthy in the patient’s past, personal or family histories except his statement that minor skin abrasions, sores and cuts frequently would become infected. The results of the initial hemogram were surprising and unanticipated, showing a hemoglobin level of 13 g/100 ml, hematocrit value 39 per cent, platelet count 282,000/mm3, reticulocyte count 1.3 per cent and a total white blood cell count of i6,000/mm3, of which 99 per cent were lymphocytes (Figure 1). Additional normal laboratory studies included fasting blood sugar, blood urea nitrogen, serum electrolytes, calcium, uric acid, creatinine, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase (LDH), alkaline phosphatase, serum bilirubin and urinalysis. The albumin was normal by serum protein electrophoresis, but diffuse hypergammaglobulinemia was present. Immunoelectrophoresis did not show a paraprotein, and the immunoglobulins, quantitated by radial immunodiffusion (Immunoplates, Hyland Laboratories Division of Travenol, Costa Mesa, Calif.) were IgG 2,160 mg/lOO ml (normal 1,120 f 250 mg/lOO ml), IgA 660 mg/lOO ml (normal 280 f 60 mg/ 100 ml) and IgM 190 mg/ 100 ml (normal 120 f 30 mg/ 100 ml). A serologic test for syphilis (VDRL), fluorescent antinuclear antibody test, Coombs’ test, heterophil antibody, tests for cryoglobulins and cryofibrinogen were all negative. The chest x-ray film was normal, although a liver-spleen scan performed with ggmT~ sulfur colloid showed both organs to be enlarged. The bone marrow could not be aspirated, but a biopsy specimen was diffusely infiltrated with lymphoid cells. The skin test with mumps antigen was positive, although there was no response to intermediate strength purified protein derivative, histoplasmosis, coccidioidomycosis and blastomycosis antigens. Sensitization to dinitrochlorobenzene (DNCB) was not performed. Because of the patient’s rapid and uneventful recovery, he was discharged to be observed as an outpatient. During the ensuing 18 months his hemoglobin level varied from 11.8 to 14.0 g/100 ml and his total white blood cell count fluctuated between 11,000 and 18,500/mm3, with 90 to 99 per cent lymphocytes. In April 1972, he again entered the hospital complaining of a sore throat and hemoptysis. A large, ulcerated, polypoid lesion was seen filling the left valecula which, by biopsy, was shown to consist of lymphocytes and plasma cells. Two weeks later the mass resolved spontaneously and could not be seen on repeat laryngoscopy. No pathogenic organisms grew in nose and throat cultures. Serum protein electrophoresis, immunoelectrophoresis and quantitative immunoglobulins were unchanged. The patient left the hospital to be kept under medical surveillance. In February 1973, after a year during which he had six different episodes of sore throats, one caused by P-hemolytic Streptococcus rapidly responding to penicillin, he was admitted to the hospital for reevaluation. In addition to repetition of the earlier studies, the results of which actually remained stable, a bone survey and a scan performed with ggmTc polyphosphate were reported as unremarkable. The bone marrow could be aspirated at this time
WITH CHROMOSOMAL
EVIDENCE OF MALIGNANCY-BRODY
ET AL.
and, in various areas, contained a mixture of lymphocytes and plasma cells (Figure 2). A paucity of immature and mature granulocytic precursors was seen, but erythroid and megakaryocyte developmental forms were adequate in number. The immunoglobulin levels again were elevated, a paraprotein was not found and isohemagglutinins were normal. The patient was referred to the Division of Hematology of the Graduate Hospital of the University of Pennsylvania and the Department of Pathology of the University of Pennsylvania School of Medicine for more detailed investigation of his circulating lymphocytes. METHODS AND MATERIALS In all the studies to be described lymphocytes were separated by differential and Ficoll-Hypaque density gradient centrifugation [2,3] when the patient’s total white cell count was 18,500/mm3, with 97 per cent lymphocytes. Formation of lymphocyte rosettes was studied using previously detailed methods [4,5]. Phagocytic cells were removed from heparinized, whole peripheral blood by a 30 minute incubation with carbonyl iron after which the blood was sedimented in 5 per cent dextran [6]. The lymphocytes then were separated as indicated earlier. Erythrocyte (RFC, E) rosettes were formed when equal volumes of washed sheep red blood cells (0.5 per cent) were mixed with lymphocytes (4 X lo6 cells/ml) in Hanks Balanced Salt Solution (HBSS) in microtiter plates (Cooke Engineering, Alexandria, Va.). These preparations then were centrifuged at 200 g for 5 minutes at 22’C, incubated for an additional hour at 22’C, gently resuspended and studied microscopically in a hemocytometer. A rosette was defined by adherence of 3 or more sheep red blood cells to a lymphocyte. EAC (complement) rosettes were prepared using sheep red blood cells incubated at 37’C for 30 minutes with 19s rabbit antibody to Forsmann antigen diluted 1: 1000 with HBSS. These sensitized cells then were kept at 37OC for 30 minutes with a 1:lO dilution of serum from normal C3H mice as a source of complement. Finally, these EAC cells (0.5 per cent suspension) were mixed in equal volumes with lymphocytes (0.1 ml), centrifuged at 200 g at 37’C, resuspended and evaluated as just outlined. To quantitate lymphocyte immunofluorescence, the cells were prepared as for rosettes and suspended in HBSS at 10 X 1O6 cells/ml. Then 0. I ml aliquots were incubated for 30 minutes at 37OC with 0.1 ml of monospecific rabbit antihuman serum immunoglobulin (G,A,M,D,E,k) components obtained commercially (Meloy, Falls Church, Va.). These preparations were washed 3 times and mounted in a 9:l glycerol:buffered saline solution medium. The slides were examined immediately using a Leitz fluorescent microscope with a Ploem attachment and results recorded as per cent positive cells. To evaluate mitogen response, cultures contained lo6 lymphocytes in 1 ml Modified Eagles Medium with 10 per cent human AB serum and 50 pg/ml phytohemagglutininM (PHA) (Difco, Detroit, Mich.). Proliferation was measured at 3, 5 and 7 days in duplicate cultures by the addition of tritiated thymidine (0.25 &i) 16 hours before termination of the culture and counting by standard scintillation methods.
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Chromosome preparations were made from 3 and 7 day cultures. Colchicine was added 3 hours before termination, and the air-dried chromosome preparations were stained with aceticorcein [7]. Finally, a recently described assay which measures the binding of 14C-alkylating agents to lymphocytes [8] also was performed as a further means of distinguishing between T and B cells. Previous and ongoing experiments in this laboratory have demonstrated that the B cells in conventional chronic lymphocytic leukemia bind less 14C-labelled alkylating compounds than their T cell counterparts [8,9]. On this basis, 12 pg 14C-cyclophosphamide (14CCTX) (speclflc actlvlty 7.6 mCl/mmol, New England Nuclear, Boston, Mass.), a concentration shown to be cytotoxlc in vitro [lo], and approximating the cllnically employed dosage [ 111, were added for 2 hours to 48 hour old, unstimulated lymphocyte cultures In TC 199 (Dlfco, Detroit, Mlch.) and 30 per cent autologous plasma. The cells were washed twice with saline solution, the entlre pellet was digested In Hyamlne (Rohm & Haas, Philadelphia, Pa.) and then transferred to vlals for counting by sclntlllation spectrometry. Mitogen stlmulatlon and 14C-CTX bindlng also was accompllshed with lymphocytes from the patient’s parents and two sisters none of whom had physical or laboratory abnormalities. RESULTS The results which follow must be evaluated in terms of absolute numbers and relative proportions of lymphocyte subgroups, both in normal as well as in the patient’s blood. It should be acknowledged that multiple surface marker assays vary within different laboratories and are not always in agreement, and that the cells under study do not necessarily retain every feature ascribed to their normal counterparts. These are the reasons additional methods of differentlation are being devised, as is true In this laboratory with the Cytoxan measurement, and as holds for the preparation of antithymocyte antiserums which, up to the present, has enjoyed limited use and availability. There were 38 per cent peripheral lymphocytes which formed RFC (E) rosettes (normal 42 to 66 per cent). With approximately 18,000 patlent lymphocytes/mm3 this represents a total of 6,800 RFC/mm3 (rounded figures), or greater than three times the maximum number expected in normal blood (2,200/ mm3), based on a total normal leukocyte count of 10,000/mm3 and 33 per cent lymphocytes. Thus, the patient has an absolute T cell lymphocytosls when compared with the normal value. However, with the RFC method, his T cells are slightly below the minimum absolute number of 7,500/mm3 (rounded figures) anticipated In his own blood. Only 1 per cent of his lymphocytes developed EAC rosettes (normal 8 to 17 per cent), with an absolute value of 180/mm3. This is just within the range of 100 to 560/mm3 for normal blood, but far below the 1,430 to 3,050/mm3 expected with this patient’s
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total lymphocyte count. Therefore, with this technic at least, his B cells are not increased. The immunofluorescence measurements showed staining with IgG 4 per cent (normal 6 to 10 per cent), IgA 2 per cent (normal 4 to 10 per cent), IgM 6 per cent (normal 6 to 14 per cent), IgD 3 per cent (normal 1 to 10 per cent), IgE 0 per cent (normal 2 to 5 per cent) and k 0 per cent (normal 7 to 24 per cent). There were 2,700/mm3 lymphocytes (rounded figures) carrying immunoglobulin receptors, somewhat above the maximum of 1,600/mm3 allowed in normal blood, but below the minimum of 3,400/mm3 for the patient’s own circulation. Since some of the positive cells may reflect adsorbed immunoglobulins from the serum [ 121, the immunofluorescence values may be spuriously high. This outcome may now be avoided by exposing the lymphocytes to trypsin, which removes the extraneous surface protein [ 131, but this was not being used at the time of this study. Nevertheless, this leaves 7,200/mm3 lymphocytes unaccounted for. The PHA cultures, a usually accepted test of T cell presence and reactivity, demonstrated a proliferation curve comparable to that seen in normal subjects: 41,000 dpm on day 3, followed by a decrease to 6,000 on day 5 and 1,300 on day 7. In contrast, lymphocytes from patients with chronic lymphocytic leukemia, now considered a B lymphocytosis, consistently show a delayed and reduced PHA response. Adequate chromosome preparations were obtained from PHA-stimulated cultures. Chromosome counts were made on a total of 100 cells: 67 from 3 day PHA cultures and 33 from 7 day PHA cultures. The majority were diploid (79 cells, 46 chromosomes; 2 cells, less than 46 chromosomes), but an aneuploid subpopulation (19 cells) with 47 chromosomes was present in both the 3 and 7 day cultures. The aneuploid cells were generally of poor technical quality, a common finding with neoplastic cells In chromosome preparations [ 141, but karyotype analysis revealed the extra chromosome to be a member of group C (chromosome 6-12 and the X, Figure 3). No other significant abnormality was noted in karyotype analyses of cells with either 46 or 47 chromosomes, the large size of the Y chromosome being considered a normal variant. The binding of 14C-CTX by lymphocytes from 13 normal donors gave a mean of 324 cpm (range 172 to 623 cpm). The normal 14C-CTX radioactivity on the patient’s lymphocytes (435 cpm) is consistent with their identification as that of a T lymphocyte population. In contrast, 14C-CTX binding of lymphocytes from 11 patients with conventional chronic lymphocytic leukemia [ 15,161 gave a mean of 76 cpm (range 17 to 109 cpm). Lymphocytes from parents and siblings were normal. Additional evidence
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[9], not detailed here, supporting the T cell’s affinity for Cytoxan, comes from membrane alteration with neuraminidase, an enzyme which removes terminal sialic acid from the cell membrane by cleaving 2,3, and 2,6 ketoglycosidic linkages of the cell surface mucopolysaccharides [ 171. This maneuver markedly decreased “C-CTX binding to normal lymphocytes and artificially converted them, in a qualified way, to a form simulating B cells, which inherently contain less membrane sialic acid than their T cell relatives [18]. It is not inappropriate to assume, therefore, that those cells previously escaping recognition have expressed their presence through mitogen response and alkylating agent binding. COMMENTS Although this patient’s clinical behavior generally has not been consistent with an overt neoplasm, the lymphocyte-infiltrated bone marrow, intermittent anemia, hepatosplenomegaly and pharyngeal lymphoid tumor furnish evidence that his lymphocytosis is more than merely a benign unremitting leukemoid reaction. Furthermore, the apparent clonal’ proliferation of lymphocytes being an abnormal karyotype strongly suggests that this is a potentially malignant condition. It is difficult to be unequivocally certain as to which lymphocyte subgroup is primarily responsible for the raised total lymphocyte count. Taken together, the various procedures described indicate an absolute increase in circulating T cells, and also a proportion of null cells which could not be characterized as T or B features with the technics used here. With newer methods this outcome, especially with normal lymphocytes, will become less frequent. The functional capacities of both the T and B cell appear to be retained in vivo, at least in part. The response to mumps antigen is evidence of T cell competence, although this may not hold for all antigens. Whereas the hyperimmunogammaglobulinemia is reasonable evidence that the plasma cells in the marrow and elsewhere are secretory, the specificity and avidity of these proteins for infectious agents have not been defined. The cytogenetic data suggest that at least a proportion of the lymphocytes responding to PHA in culture (presumably T cells) are neoplastic, as indicated by the aneuploid cell line. The poor technical quality of the aneuploid metaphases, as compared with diploid cells on the same slides, argues for their neoplastic character [ 141 and probably also resulted in an underestimation of their frequency. It is possible, _ The term clonal is used in the cytogenetic sense and refers to a population of ceils with the same karyotype, presumably descendant from a single precursor. l
Figure from 3 of 100 somes,
3. Karyotype wtih extra c’ group chromosome day PHA-stimulated lymphocyte culture. Nineteen metaphases from PHA cultures had 47 chromosuggesting a neoplastic clone of T ceils.
of course, that this 47, XY, C+ clone reflects constitutional mosaicism (e.g., an XXY subpopulation) rather than a neoplastic process, but the absence of any stigmata of Klinefelter’s syndrome and the different quality of these cells from the diploid population make this conclusion seem very unlikely. If adequate preparations can be obtained from this patient in the future, autoradiographic and banding methods will permit precise identification of the extra C group chromosome and should definitely resolve this question. The lifelong history of infection provides a speculative explanation for the events described and the laboratory results obtained. It is conceivable that, initially, a general defect existed in leukopoiesis, such that abnormal differentiation of the totipotent stem cell led to persistent granulocytopenia associated with or followed by a distorted mechanism regulating the number of circulating lymphocytes [ 191. Detection of the chromosomal aberration at this patient’s present age and point in disease may make this patient especially unique in the sense that he could be a clinical prototype for patients with other lymphoreticular tumors which also may begin in a clonal fashion but are not always recognized as such because of a more subtle cellular change not measurable by present methods. Whether, as is applicaApril 1975 The AmericanJournalof Medicine Volume58
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ble in this instance, too, these clones consistently become overtly destructive may depend on the integrity of host factors such as immunosurveillance, and on any further alterations which may occur in the abnormal cell group.
ET AL.
ACKNOWLEDGMENT Grateful thanks are extended to Mrs. Jane Kohut, Mrs. Mary Haidar and the Misses Julie Jensen and Paula Henry for their technical assistance.
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Aisenberg AC, Bloch KJ: lmmunoglobulins on the surface of neoplastic lymphocytes. N Engl J Med 287: 272, 1972. Brody JI, Beizer LH: Alteration. of blood group antigens in leukemic lymphocytes. J Clin Invest 44: 1582, 1965. Bdyum A: Separation of leukocytes from blood and bone marrow: with special reference to factors which influence and modify sedimentation properties of hematopoietic cells. Stand J Clin Lab Invest 21 (suppl 97): 1, 1968. Medes NF, Tolnai MEA, Silveria NPA, Gilbertson RB, Metzgar RS: Detection and specific depletion of subpopulations of human lymphocytes by rosette formation with antibody-complement sensitized erythrocytes and unsensitized erythrocytes. J lmmunol 111: 860, 1973. Daniele RP, Rowlands DT Jr: In preparation. van Rood JJ, van Leeuwen A, Zweerus R: 4a and 4b antigen, do they or don’t they? Histocompatibility Testing (Terasaki P, ed), Copenhagen, Munksgaard. 1970, p 93. Moorehead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA: Chromosome preparations of leucocytes cultured from human peripheral blood. Exp Cell Res 20: 613, 1960. Brody JI: Diminished binding of alkylating agents by lymphocytes in chronic lymphocytic leukemia. J Clin Invest 52: 13a, 1973. Brody JI: Differential uptake of alkylating agents by normal and leukemic lymphocytes. Cancer (in press). Singer BL, Brody JI: In vitro activation of cyclophosphamide
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