Prolymphocyte Leukemia with IgM Hypogammaglobulinemia

Prolymphocyte Leukemia with IgM Hypogammaglobulinemia TORU TAKENAKA, M.D., HIROKAZU NAKAMINE, M.D., TORU NISHIHARA, M.D., TADAAKI TSUDA, M.D., MASATO TSUJIMOTO, M.D., AND JIRO MAEDA, M.D.

Departments of Laboratory Medicine, Pathology, and Internal Medicine, Wakayama Medical School, Wakayama, Japan

A case of prolymphocyte leukemia with IgM hypogammaglobulinemia in a 47-year-old man was presented. The leukemic cells possessed la-like antigen and receptors for the third component of complement but lacked surface immunoglobulins, cytoplasmic IgM, receptors for sheep red blood cells, or terminal deoxynucleotidyl transferase activity. In vitro immunoglobulin production experiments demonstrated that the leukemic cells did not have the capacity to produce IgM, while patient's T cells were shown to possess helper function on normal B cells to produce immunoglobulins. By these findings, together with the presence of selective IgM hypogammaglobulinemia, it was suggested that the leukemic cells were derived from a B-cell clone of a stage in differentiation and maturation of IgM-forming B-cell spectrum. (Key words: Prolymphocytic leukemia; IgM hypogammaglobulinemia; la-like antigen) Am J Clin Pathol 1983; 80: 237-242

Report of a Case

Received October 25, 1982; received revised manuscript and accepted for publication January 5, 1983. Address reprint requests to Dr. Nakamine: Department of Laboratory Medicine, Wakayama Medical School, 7-Bancho, Wakayama City 640, Wakayama, Japan.

Materials and Methods Morphologic and Cytochemical Examinations of the Leukemic Cells Peripheral blood smears were stained with MayGriinwald and Giemsa, and cytochemically stained for peroxidase, a-naphthyl butyrate esterase, and acid phosphatase. For electron microscopic examinations, mononuclear cells were isolated from heparinized peripheral blood by Ficoll-Isopaque® (Pharmacia Fine Chemicals,

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IN 1974, Galton and co-workers7 proposed a new clinicopathologic entity of prolymphocytic leukemia (PLL), which was characterized by increased prolymphocytes in peripheral blood and massive splenomegaly with little lymphadenopathy. Whereas PLL can be distinguished from typical chronic lymphocytic leukemia (CLL) in clinicopathologic features, it is generally considered to be a variant of CLL.6 Recent advances in immunology have made it possible to search for the origin of lymphoid leukemic cells, and it has been confirmed that both T- and B-cell types of PLL exist as in the case of CLL.41819 Moreover, PLL, which expressed helper T-cell phenotype on the surface of the leukemic cells was reported recently.5 In the present report, a case of PLL with selective IgM hypogammaglobulinemia was presented. Leukemic cells of the case were confirmed to possess la-like antigen and receptors for the third component of complement (C3) but did not bear surface immunoglobulins (slg), cytoplasmic IgM (c-IgM), or receptors for sheep red blood cells. Some immunologic studies were performed to characterize the origin of the leukemic cells and to determine whether there was any relationship between IgM hypogammaglobulinemia and a defect of slg on the leukemic cells.

A 46-year-old Japanese man visited his physician at home because of tenderness and swelling at left hypochondrium in December 1980. Massive splenomegaly up to 4 cm below the umbilicus was found, and he was transferred to our hospital. He had a duodenal ulcer at the age of 40. Physical examination revealed severe conjunctival anemia and lymphadenopathy at the cervical, submandibular, axillary, and inguinal regions. Enlarged lymph nodes were soft and movable with no tenderness. The spleen was enlarged to 35 cm below the left costal margin, the liver was felt 4 cm below the right costal margin. The hemoglobin was 8.9 g/dL. The leukocyte count was 181,000/ nL with 90.4% leukemic cells (prolymphocytes) and 8% normally appearing small lymphocytes. The platelet count was 124,000/^L, and prothrombin and partial thromboplastin time were normal. Serum iron level was 57, U1BC was 262, and TIBC was 316 Mg/dL. Serum hepatic function tests were normal, except alkaline phosphatase and lactate dehydrogenase, which were elevated to 186 and 477 U/L, respectively. Renal function and serum electrolyte levels were normal. Total serum protein was 7.6 g/dL, and each immunoglobulin level was IgG, 1,520 mg/dL; IgA, 437 mg/dL; IgM, 40 mg/dL; IgD, 6 mg/ dL; and IgE less than 25 IU/mL. Muramidase level in serum was 8.9 mg/dL and was not detected in urine. Serologic examinations such as cold hemagglutinin, Coombs test, and thyroglobulin test had negative results, while CRP and RA test had positive results. Skin tests with purified protein derivative and 0.1% dinitrochlorobenzene showed 13 X 18 and 11X13 mm of wheal, respectively. Scintillation scanogram using "°Tc demonstrated massive splenomegaly. Bone marrow aspiration was unsuccessful (dry tap), in spite of two attempts. Following administration of cyclophosphamide and vincristine, the patient's general condition improved with a decrease of leukocyte count and slight reduction of the splenic size. However, serum IgM level was persistently low (8-40 mg/dL) during his stay in the hospital. His condition now is being followed on an outpatient basis.

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Uppsala, Sweden) density gradients. They were fixed in 1% glutaraldehyde, postfixed in 1% osmium tetroxide, and then applied for scanning and transmission electron microscopic studies. A biopsy was done on a cervical lymph node; the specimen was fixed in 10% formalin or 1% osmium tetroxide, and examined with light and electron microscopes. Marker Studies of the Leukemic Cells

IgG and IgM Production In Vitro According to Moretta and associates,14 peripheral blood mononuclear cells from the patient (P) and normal individuals (N) were separated into T and non-T cell populations. Then, 1 X 105 T and/or 5 X 104 nonT cells from the patient (P T cells and/or P non-T cells) were co-cultured with 1 X 105 T and/or 5 X 104 non-T cells from five normal individuals (N T cells or N nonT cells, or both) for seven days in the presence of PWM (Grand Island Biological Co., New York, NY) at a final concentration of 1 Mg/mL." IgG and IgM levels in the media were measured by radioimmunoassay.813 Briefly, rabbit antibodies specific for human 7-chains and Mchains (Behringwerke) were attached onto CNBr-activated Sepharose beads (Pharmacia Fine Chemicals). The antibody-attached Sepharose beads were incubated with co-cultured medium for 24 hours, spun down, and washed with PBS. Then, 125I-labeled human IgG or IgM (New England Nuclear, Boston, MA) were added and incubated for 18 hours. Radioactivity on the Sepharose beads was counted with a scintillation counter, and im-

munoglobulin levels were estimated from the standard curves. DNA Synthesis of Mononuclear Cells In Vitro Mononuclear cells, T cells, and non-T cells, isolated as described above, were adjusted to 1 X 106/mL of RPMI 1640 medium supplemented with 20% fetal calf serum. DNA synthesis was examined as described previously15 with minor modifications. PHA (PHA-P, Difco Laboratories, Detroit, MI), PWM and Con A (Pharmacia Fine Chemicals) were used at final concentrations of 100, 10, and 4 jug/mL, respectively. Results Morphologic and Cytochemical Examinations of the Leukemic Cells As shown in Figure 1 (inset), the leukemic cells in peripheral blood were larger than those of typical CLL, and their diameter varied 14-18 nm. The nuclei were round, some of which had one or two fine nucleoli. The cytoplasm was moderately abundant and stained pale blue with May-Grunwald and Giemsa. In cytochemical preparations, the cytoplasm of the leukemic cells was not stained for peroxidase or a-naphthyl butyrate esterase but stained for acid phosphatase, which was reduced after treatment of tartrate. Under transmission electron microscopic study, the nuclei of the leukemic cells were round without convolution or cleavage, and tight attachment of aggregated chromatin to the nuclear rim was observed. Mitochondria were relatively abundant with minimal endoplasmic reticulum, and neither parallel tubular array (ribosome-lamella complex)10 nor inclusions' were found in the cytoplasm (Fig. 1). Scanning electron microscopic study revealed the scattering of short microvilli on the surface of numerous cells (Fig. 2). The biopsied lymph node wasfilledwith monotonous large lymphoid cells, and normal architecture was effaced (Fig. 3). The capsule of the node was partially destroyed, and large lymphoid cells infiltrated into the surrounding connective tissue. The ultrastructural fea-

FlG. 1 (upper). Transmission electron micrograph of the leukemic cells in peripheral blood. The nuclei showed tight attachment of aggregated chromatin to the nuclear rim with prominent nucleoli (n). The cytoplasm contained minimal endoplasmic reticulum, scattered mitochondria (m); and vacuoles (va). Uranyl acetate and lead nitrate (X6.900). Inset. Photomicrograph of peripheral blood smear. The leukemic cells were large (14-18 /im in diameter) and had abundant cytoplasm with some vacuoles. One or two fine nucleoli occasionally were seen. May-Grilnwald and Giemsa (X 1,800). FIG. 2 (lower, left). Scanning electron micrograph of the leukemic cells in peripheral blood. Short microvilli were scattered on the cell surface (X2.200). FIG. 3 (lower, right). Photomicrograph of a biopsied cervical lymph node. Normal architecture of the lymph node was effaced by diffuse proliferation of the leukemic cells. Hematoxylin and eosin (X340).


Rosette formation with sheep red blood cells or IgMC3-coated bovine red blood cells (E or EAC rosette) was performed by conventional methods. SIg were examined using FITC-conjugated F(ab')2 fragments of goat IgG against human immunoglobulins (Cappel, Cochranville, PA) after incubation of mononuclear cells at 37°C for 18 hours. C-IgM was examined using acetone-fixed films with FITC-conjugated rabbit antibody against human /t-chain (Behringwerke, Marburg, West Germany). Peripheral blood smears were studied for terminal deoxynucleotidyl transferase (TdT) activity9 with TdT kit (Bethesda Research Laboratories; Gaithersburg, MD).

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tures of large lymphoid cells in the node was similar to those of the leukemic cells in peripheral blood. Desmosome structure or digital projections were not observed in intercellular space. Marker Studies of the Leukemic Cells The percentage of E rosette was less than 5%, and the leukemic cells did not form E rosette on cytocentrifuge preparations. TdT activity was not detected. Neither slg nor c-IgM were detected in significant percentages, but more than 50% of the cells formed EAC rosette. Ia-like antigen was demonstrated on the leukemic cells by Dr. Machii (Osaka University).

icant (P < 0.05). While IgM was not detected (less than 5 ng/mL) in the allogeneic co-culture of N T cells and P non-T cells, 23 ± 20 ng/mL of IgG was detected (experiment 4). Small amounts of IgG and IgM were detected in the allogeneic co-culture of N non-T cells and P non-T cells (experiment 5). Immunoglobulin production in the autologous co-culture of N T cells and N non-T cells was not affected significantly by either P T cells or P non-T cells (experiments 6 and 7). Small amounts of IgG and IgM were detected in the culture of N non-T cells alone, but immunoglobulins were not detected in the culture of P non-T cells alone (experiments 8 and 9).

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The results of immunoglobulin production experiments were shown in Figure 4. When P T cells were cultured with PWM-stimulated P non-T cells (mainly composed of the leukemic cells), the amounts of IgG and IgM synthesized were less than 5 ng/mL, while autologous co-culture of N T cells and N non-T cells synthesized 2,216 ± 662 ng/mL of IgG and 4,243 ± 973 ng/mL of IgM (experiment 1 and 2). Whereas the amounts of synthesized immunoglobulins were slightly reduced in the allogeneic co-culture of P T cells and N non-T cells (experiment 3) in comparison with those in experiment 2, these values were not statistically signif-

In-vitro DNA synthesis experiments were performed three times in conjunction with morphologic and lymphocyte marker studies. Responsiveness of peripheral blood mononuclear cells to mitogens, especially to PHA, was preserved comparatively, despite the fact that most of the cells were morphologically leukemic (Fig. 5). When mononuclear cells were separated into T and nonT cells, T cells showed significant incorporations of 3 H-TdR in the presence of mitogens, which was almost comparative to those of unseparated mononuclear cells. Non-T cells, on the other hand, did not show any response to mitogens.


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FIG. 5. DNA synthesis of mononuclear cells, T cells, and non-T cells in peripheral blood to mitogens. Leukocyte count of the blood sample was 88,400/ML with 80% leukemic cells. Counts per minute (cpm) of control cultures (saline instead of mitogens) were less than 300. Cpm of peripheral blood mononuclear cells from 41 healthy adults were 925 ± 150 (saline), 39,593 ± 4,376 (PHA), 17,191 ± 2,015 (PWM), and 29,565 ± 5,259 (Con A).

The present case was considered to fulfill the criteria for PLL in its clinical features7 and morphologic findings.12 In lymphocyte marker studies, it was demonstrated that the leukemic cells possessed la-like antigen and receptors for C3 but lacked sig, c-IgM, receptors for sheep red blood cells, or TdT activity. These results indicated that the leukemic cells were not derived from a T-cell clone. The discrepancy of surface markers (positive la-like antigen and C3 receptors, negative sig) occasionally were observed on some neoplastic lymphoid cells.2,3 By the simultaneous occurrence of the defect of sig on the leukemic cells and IgM hypogammaglobulinemia, it was suggested that the leukemic cells would be derived from a B-cell clone. As shown in immunoglobulin production experiments, the leukemic cells did not have the ability to produce IgM nor to help or suppress immunoglobulin production by normal B cells. Moreover, patient's T cells were shown to possess helper function on norrrial B cells to produce immunoglobulins. Thus, it strongly was suggested that IgM hypogammaglobulinemia would result from the impairment of IgM-forming B-cell spectrum but not of T-cell populations. And it is reasonable to draw the conclusion that the leukemic cells were derived from a committed B-cell clone of a stage in differentiation and maturation of IgM-forming B-cell spectrum. On the other hand, surface IgM-bearing B cells that had the capability to differentiate into IgG- and IgAforming B cells might be intact, because serum levels of them were normal. An interesting phenomenon observed in the experiments of in-vitro DNA synthesis was that unseparated

mononuclear cells (1 X 106/mL) composed of more than 80% leukemic cells responded well to PHA and PWM. After separation, T cells (1 X 106/mL) still responded well to mitogens, while non-T cells, most of which were composed of the leukemic cells, did not show any response. It was unlikely that the responsiveness of unseparated mononuclear cells were derived from T cells, because the amount of T cells in unseparated cells was too small to show significant responsiveness. Therefore, as reported recently,16,17 it is possible to consider that the leukemic cells might become responsive to polyclonal T- and B-cell activators via mitogen-indiiced T cell factor(s). However, further cases should be examined before making a conclusion that the behavior is one of the common properties of PLL. Acknowledgment. The authors thank Dr. T. Machii (Division of Internal Medicine, Department of Clinical Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) for the detection of la-like antigen on the leukemic cells.

References 1. Appay M-D, Bariety J, Bretton R: Characterization by immunoperoxidase of the lymphocytes with bundle-shaped tubular (BST) inclusions in human normal peripheral blood. Blood 1980; 55:67-70 2. Brouet J-C, Flandrin G, Sasportes M, Preud'homme JL, Seligmann M: Chronic lymphocytic leukemia of T-cell origin: Immunological and clinical evaluation of 11 patients. Lancet 1975;890-894 3. Brouet J-C, Prieur A-M: Membrane markers on chronic lymphocytic leukemia cells: B cell leukemia with rosettes due to antisheep erythrocyte antibody activity of the membrane bound IgM and a T-cell leukemia with surface lg. Clin Immunol Immunopathol 1974; 2:481-487 4. Catovsky D, Galetto J, Okos A, Galton DAG, Wiltshow E, Staphopoulos G: Prolymphocyte leukemia of B and T cell type. Lancet 1973; 232-234


Discussion

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12. Kjeldsberg CR, Bearman RM, Rappaport H: Prolymphocytic leukemia: An ultrastructural study. Am J Clin Pathol 1980; 73:150-159 13. McLachlan SM, Smith Br, Hall R: Kinetics of immunoglobulin production by cultured human peripheral blood lymphocytes. J Immunol Methods 1978; 21:211-216 14. Moretta L, Webb SR, Grossi CE, Lydyard PM, Cooper MD: Functional analysis of two human T-cell subpopulations: Help and suppression of B-cell responses by T-cells bearing receptors for IgM or IgG. J Exp Med 1977; 146:184-200 15. Nakamine H, Nishihara T, Saito.K, Takenaka T, Maeda J: Needle-shaped inclusions in plasma cells in a patient with hypogammaglobulinemia. Am J Clin Pathol 1982; 78:549-555 16. Robert K-H: PHA-induced soluble factors) can activate B-celis from patients with chronic lymphatic leukaemia. Clin Exp Immunol 1979;37:517-522 17. Robert K-H: Induction of monoclonal antibody synthesis in malignant human B cells by polyclonal B cell activators: Relationship between B cell subsets and prognosis. Immunol Rev 1979;48:123-143 18. Sibbald R, Catovsky D: Complete remission in prolymphocytic leukemia with the combination chemotherapy-CHOP. Br J Haematol 1979;42:488-490 19. Woessner S, Lafuente R, Sans-Sabrafen J, Vives J, Rozman C: Prolymphocytic leukemia of T-cell type: Immunological, enzymatic and ultrastructural morphometric characteristics. Br J Haematol 1978; 39:9-15

Central Nervous System Involvement as a Presenting Feature of Acute Monocytic Leukemia: Preceding Leukemia by 12 Months JAMES E. HAMOUS, M.D., LINDA E. ANSBACHER, M.D., JAMES O. ARMITAGE, M.D., AND FRED R. DICK, M.D.

This report describes a patient who presented with a diffuse malignant central nervous system (CNS) process composed of cells with monocytic differentiation one year before the development of acute monocytic leukemia. A similar presentation has been documented for acute lymphocytic leukemia, but to the best of our knowledge, this is the first report to detail a diffuse CNS malignancy as the presenting symptom for acute monocytic leukemia (Key words: Monocytic leukemia; Central nervous system; Cytospin) Am J Clin Pathol 1983; 80: 242245 ACUTE MONOCYTIC LEUKEMIA now is considered to be a malignancy derived from bone marrow stem cells and has been included by the French American

Received November 1, 1982, received revised manuscript and accepted for publication January 3, 1983. Address reprint requests to Dr. Hamous: Medical Research Center 104, Department of Pathology, University of Iowa, Iowa City, Iowa 52242.

Departments of Pathology and Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa

British Cooperative Group (FAB) as one of the six subtypes of acute nonlymphocytic leukemia, M 5 subtype. Monocytic leukemias have a well-described propensity to develop extramedullary infiltrates, primarily involving the skin and gingiva. Several recent reports also have emphasized that extramedullary masses or infiltrates may be the presenting manifestation of acute monocytic leukemia. In this article we present a case of acute monocytic leukemia in which the diagnosis of central nervous system (CNS) involvement preceded the appearance of typical acute monocytic leukemia in the bone marrow by 12 months. Such an extramedullary presentation of acute monocytic leukemia can be confused with other large cell malignancies if technics to identify the

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5. Chan WC, Check IJ, Heflher LT, Gordon D, Whitsett C, Brynes RK: Prolymphocytic leukemia of helper cell phenotype: Report of a case and review of the scientific literature. Am J Clin Pathol 1982; 77:643-647 6. Christensson B, Biberfeld G, Biberfeld P: Immunoglobulin expression in B-cell leukemias and B-cell lymphoma. Malignant lymphoproliferative disorders. Edited by Van den Tweel JG, Taylor CR, Bosnian FT. Boston, Leiden University Press, 1980, pp 229-244 7. Galton DAG, Goldman JM, Wiltshow E. Catovsky D, Henry K, Goldberg GJ: Prolymphocytic leukemia. Br J Haematol 1974; 27:7-23 8. Hirano T, Kuritani T, ICishimoto T, Yamamura Y: In vitro immune response of human peripheral lymphocytes. I. The mechanism^) involved in T cell helper functions in the pokeweed mitogen-induced differentiation and proliferation of B cells. J Immunol 1977; 119:1235-1241 9. Janossy G, Bollum FJ, Bradstock KF, McMickael A, Rapson N, Greaves MF: Terminal transferase-positive human bone marrow cells exhibit the antigenic phenotype of common acute lymphoblastic leukemia. J Immunol 1979; 123:1525-1529 10. Katayama I, Li CY, Yam U: Ultrastructural characteristics of the hairy cells of leukemic reticuloendotheliosis. Am J Pathol 1972; 67:361-366 11. Keightley RG, Cooper MD, Lawton AR: The T cell dependence of B cell differentiation induced by pokeweed mitogen. J Immunol 1976; 117:1538-1544
