Successful treatment of hairy cell leukemia with pentostatin

A 45-year-old woman was admitted to our hospital in August, 1999. Laboratory data showed a white blood cell count of 5,050/microliter with 78% abnormal lymphocytes, hemoglobin 6.8 g/dl, platelets 4.8 x 10(4)/microliter, and soluble IL-2 receptor 97,600/ml. The abnormal cells were characterized by a hairy appearance under phase contrast microscopy, and showed strong tartrate-resistant acid phosphatase activity. Immunophenotype analysis revealed that these cells were positive for CD11c, CD19 and CD25, and negative for CD5. Bone marrow biopsy showed diffuse proliferation of hairy cells with moderate myelofibrosis. We diagnosed the patient as having European-American-type hairy cell leukemia. Pentostatin was administered at a dose of 5 mg/m2 weekly. After twelve doses, the peripheral blood data returned to the normal range with no hairy cells in the blood or bone marrow, although slight splenomegaly remained. The patient underwent splenectomy in December of the same year, and we were unable to find any hairy cells by histological and immunohistochemical examination. Although most patients with hairy cell leukemia in Japan have the Japanese variant, and the European-American type is rare, pentostatin is as effective as it is for European and American patients.     

Distinct subtype within the spectrum of hairy cell leukemia.

Most cases of hairy cell leukemia represent malignancies of B cells. However, recent findings suggest that there is a spectrum of functional capacities within the entity hairy cell leukemia. Two patients with hairy cell leukemia, whose malignant cells in the peripheral blood showed both T- and B-cell features, are reported. The malignant cells of the spleens showed only B-cell characteristics. The hairy cells of both patients did not adhere to glass and lacked the la antigen. Both patients showed pronounced polyclonal hypergammaglobulinemia and developed frank leukemic blood pictures after splenectomy. Within the spectrum of hairy cell leukemia, these two cases probably represent a distinct subtype.     

An antigen common to chronic lymphocytic and hairy cell leukemia cells not shared by normal lymphocytes or by other leukemic cells

Rabbit xenoantisera and mouse monoclonal antibodies prepared against human chronic lymphocytic leukemia (CLL) B cells were found to react against a single polypeptide chain with a mol wt of 69 kd found on leukemic cells of all CLL (N = 40) and B type hairy cell leukemia (HCL) patients (N = 9) examined. This common CLL-associated antigen (cCLLa) was not detectable on circulating T or B lymphocytes, thymocytes, lymph node and splenic lymphocytes, or bone marrow leukocytes from normal persons. In addition, the cCLLa was not detectable on cultured T or B lymphoblastoid cell lines or on malignant cells from other forms of lymphocytic or myelocytic leukemia. Non-Hodgkin's lymphoma cells also failed to express the antigen. Autologous cultured lymphoblastoid cell lines were established from residual normal B cells from a CLL patient whose cells were used to generate one of the antisera. Absorption of the antibody with these cultured polyclonal B cells did not affect the anti-CLL activity, which suggests that the cCLLa is not HLA related. Unlike the T cell differentiation complex gp65-71, the cCLLa was not expressed on fetal or cord blood lymphocytes or on mitogen-stimulated normal lymphocytes and was distinct from the antigen recognized by the LEU-1 antibody in spite of their similar mol wt. The cCLLa was also determined to be unrelated to the human T cell leukemia lymphoma virus (HTLV-1). One of the monoclonal antibodies generated against the cCLLa was a complement binding IgG which exhibited highly selective cytotoxic activity against 100% of cells bearing the cCLLa. Such an antibody might prove clinically useful in early diagnosis and treatment of CLL and HCL.     

Inhibition of leukemic cell proliferation by factor(s) released from irradiated lymphocytes of B-chronic lymphocytic leukemia patients

An attempt was made to clarify the mechanism by which splenic irradiation in patients with B-chronic lymphocytic leukemia (B-CLL) can induce a reduction in lymph node size. For this purpose peripheral blood lymphocytes from B-CLL patients were exposed to cobalt irradiation and were cultured for 1–8 days. The effect of the supernatants on the proliferation capacity of normal and malignant human cells was examined. A suppression of phytohemagglutinin (PHA)-induced proliferation of autologous and heterologous B-CLL lymphocytes was observed, whereas there was no effect on the proliferation of lymphocytes obtained from healthy volunteers. in addition, supernatants of irradiated B-CLL lymphocytes inhibited thymidine incorporation into blasts derived from patients with acute leukemia and the lymphoblastoid cell line Daudi, but they did not exert any effect on normal cells obtained from human embryonic liver. These results suggest the secretion of some factor(s) by irradiated B-CLL lymphocytes, which may inhibit the proliferation of malignant cells but has no effect on normal cells. © 1994 Wiley-Liss, Inc.     

Hairy cell leukemia: functional, immunologic, kinetic, and ultrastructural characterization.

A diagnosis of hairy cell leukemia was made by optic microscopy, phase-contrast microscopy, electron microscopy, scanning microscopy, and histochemistry of the abnormal blood cells. In vivo these cells were found to have a half-time in the blood of approximately 150 hr. In vitro they had the capacity to adhere firmly to plastic, making it possible to obtain a pure population of hairy cells. Neither T-rosette formation nor phytohemagglutinin (PHA) transformation could be demonstrated in these cells. On the other hand, the presence of immunoglobulins on the surface of the hairy cells (HC) by immunofluorescence, and the synthesis and secretion by these cells of IgM type lambda-chains shown by radioimmunodiffusion, were in favor of their B-type lymphocyte origin. Similarities to chronic lymphocytic leukemia were apparent.     

Antigenic phenotype of splenic hairy cells

Hairy cell leukemia, a distinct clinical and morphologic lymphoproliferative disorder, is characterized by the proliferation of mononuclear cells of uncertain derivation. Attempts to identify the cell of origin have used studies either of functional capabilities or of membrane/cytoplasmic antigens. Only a few cases have been studied via monoclonal antibodies. Frozen sections of splenic tissue involved with hairy cell leukemia were studied with a variety of monoclonal antibodies having specificity for differentiation antigens using the avidin-biotinylated peroxidase complex technique. Conventional direct and indirect immunohistochemical study was used for immunoglobulin heavy and light chains. In all but one case, the neoplastic cells expressed monoclonal immunoglobulin. Although T cells were identified in persisting periarteriolar sheaths and occasionally admixed with red blood cells in pseudosinuses, phenotypic expression of intrathymic or peripheral T cell antigens by the proliferating neoplastic cells was not observed. Conversely, expression of B1 and HLA-Dr antigens by splenic hairy cells was documented in all 10 cases. Hairy cell leukemia cells did not express either monocyte antigens (M1 and MO2) or the antigens expressed by early (J5) and intermediate (B2) B cells or plasmacytoid lymphocytes and plasma cells (T10). These immunohistochemical results with monoclonal antibodies provide further evidence that hairy cell leukemia is characterized by a combination of antigens peculiar to mature B lymphocytes.     

A hairy B cell lymphoproliferative disorder resembling hairy cell leukemia

This case report describes a hairy B cell lymphoproliferative disorder (HBLD) with clinical and hematological features resembling hairy cell leukemia. The patient was a 29-year-old female who demonstrated atypical lymphocytes in her peripheral blood. Physical examination demonstrated splenomegaly, but there were no palpable superficial lymph nodes. Hematological examination showed a leukocyte count of 10.6 x 10(3)/mm3 with 41% atypical lymphocytes. Bone marrow examination showed a normal cellular and an atypical lymphocyte count of 42%. The atypical lymphocytes had microvilli and prominent membranous ruffles on their surfaces. Atypical lymphocytes expressed CD5- CD10- CD11c+ CD19+ CD20+ CD23- CD25- on the surface of the cells on examination by with a fluorescence activated cell sorter. Although these findings were similar to hairy cell leukemia, Japanese variant, the surface marker of the kappa chain and lambda chain was unbiased and studies of immunoglobulin gene rearrangements and expression showed polyclonal proliferation of B cells. Therefore, we diagnosed this patient as having HBLD. Because she did not demonstrate anemia or thrombocytopenia, she is not currently receiving medication. To date, the atypical lymphocyte count has not changed.     

Characterization of the phytohemagglutinin-induced proliferating lymphocyte subpopulations in chronic lymphocytic leukemia patients using a clonogenic agar technique and monoclonal antibodies

Peripheral blood lymphocytes from normal donors and patients with chronic lymphocytic leukemia, B-cell type, were purified into T, helper T, and suppressor T lymphocytes by fluorescence-activated cell sorting using OKT3, OKT4, and OKT8 monoclonal antibodies. The maximum response of the purified subpopulations to stimulation by phytohemagglutinin (PHA) was determined by measuring the production of colonies when the stimulated cells were grown on agar. The helper T cells in normal and CLL patients were the most responsive to PHA stimulation, although the responsiveness of helper T cells to PHA was decreased in CLL. Purified CLL B cells responded minimally to PHA stimulation, but normal B lymphocytes did not. The abnormal response of CLL lymphocytes to PHA appears to be due abnormal helper T cells, and, to a smaller extent, to the ability of CLL B lymphocytes to respond.     

Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in hairy cell leukemia.

In vitro 14-day cultures of peripheral blood mononuclear cells from hairy cell leukemia patients consistently showed the presence of hematopoietic stem cells giving rise to multilineage colonies containing a high proportion of lymphoid cells associated with the myeloid and erythroid progenitors. These stem cells are not the hairy cells but appear to be pluripotent lymphomyeloid primitive stem cells persisting in this leukemia. Interferon alpha c or beta 1 did not inhibit the growth of these colonies, as they did the growth of colonies of normal hematopoietic progenitors, but markedly decreased the ratio of lymphoid to myelomonocytic cells, by increasing the formation of monocytes and other nonlymphoid cell types in these multilineage colonies. Interferon gamma did not have the same effects on differentiation.     

Host defense deficiency in hairy cell leukemia and its correction by leukocyte transfusion

A similar defect host defense mechanisms in hairy cell leukemia was defined in two patients. Surface-adherent monocytes were not detected in the peripheral blood nor were monocytes that mediate antibody- dependent cell-mediated cytotoxicity (ADCC) to isoantibody-coated human erythrocytes. In addition, lymphocytes of both patients failed to show blastogenic responses to concanavalin A (Con-A) and pokeweed mitogen (PWM) but showed a vigorous response to phytohemagglutinin (PHA). Other immunologic abnormalities were present but were either moderate in degree or were not present in both patients. In vitro lymphocyte blastogenic responses were fully restored by incubation of patients' leukocytes with a normal donor's adherent monocytes. One patient received daily allogeneic leukocyte transfusion for 4 days. This resulted in complete normalization of monocyte adherence and ADCC that persisted for several months after transfusion and was associated with hemotalogic improvement. Therapy in case 1 resulted in correction of the blastogenic responses to Con-A and PWM. Thus, a host defense defect in hairy cell leukemia has been defined in 2 patients and a preliminary result suggests that therapy with leukocyte transfusions may be useful in the postsplenectomy patient with an infectious complication and should be explored further.     

Interaction of peanut agglutinin with normal human lymphocytes and with leukemic cells.

The interaction of peanut agglutinin (PNA) with human thymocytes, peripheral blood lymphocytes, and peripheral blood cells of various types of leukemia was investigated by using fluorescein isothiocyanate-conjugated PNA. The majority of human thymocytes (60-80%) bind the lectin. The major subpopulation of thymocytes that is PNA-positive was separated from the PNA-negative cells by differential agglutination with the lectin. The two thymocyte subpopulations were tested in the mixed lymphocyte reaction and with the phytohemagglutinin of Phaseolus vulgaris. The poor response of the PNA-positive thymocytes to these stimuli indicates that these thymocytes are functionally immature. The fluorescein isothiocyanate-PNA-binding test with peripheral blood lymphocytes of leukemic patients revealed that in most acute leukemias the PNA receptor is exposed on the blastic cells, whereas in most cases of chronic leukemia the peripheral blood lymphocytes are PNA-negative. The validity of PNA as a marker of immature blood cells and its potential clinical application are discussed.     

Proliferation, differentiation, and cytogenetics of chronic leukemic B lymphocytes cultured with mitomycin-treated normal cells

Lymphocytes from 6 patients with chronic lymphocytic leukemia of the B- cell variety (B-CLL) were cultured with equal numbers of mitomycin- treated mononuclear cells from normal blood. When stimulated with pokeweed mitogen (PWM), phytohemagglutinin (PHA), or the tumor- promoting agent, phorbol tetradecanoyl-acetate (TPA), the CLL cells proliferated actively by day 3 or 4 of culture, and in four cases, differentiated to significant numbers of immunoglobulin-containing cells. Chromosome studies on the proliferating lymphocytes demonstrated a cytogenetically abnormal clone in three patients, including two with a 14q+ marker chromosome and two with a translocation involving the short arm of chromosome 9. One patient had a translocation from 22q to 14q, producing a Philadelphia chromosome as well as the 14q+ marker. The results indicate that the neoplastic lymphocytes of B-CLL may proliferate and differentiate when appropriately stimulated in vitro, and that chromosomally abnormal clones are not uncommon. With several techniques now available for successful short-term culture of B-CLL lymphocytes, there is opportunity for better understanding of the cellular alterations in this disease.     

Growth hormone stimulation of normal and leukemic human T-lymphocyte proliferation in vitro

In order to investigate the effect of human growth hormone on T lymphocytes, we utilized a clonogenic assay for mitogen-responsive human peripheral blood T lymphocytes. Lymphocytes were purified by density gradient centrifugation and incubated in the presence of phytohemagglutinin using a two-layer agar technique for CFU- T- lymphocyte culture. Nanogram concentrations of human growth hormone, ovine prolactin, human chorionic somatomammotropin, or growth hormone fragment were added to cell cultures. Growth hormone potentiated normal T-cell colony formation in a species-specific manner. Cells from a homogeneous T-lymphoblast line derived from a patient with a T-cell variant of hairy cell leukemia also showed augmentation of colony growth in the presence of human growth hormone. These studies provide evidence for a direct effect of growth hormone on normal and some neoplastic human T cells.     

Lipopolysaccharide responsiveness of malignant lymphoid cells in a patient with hairy cell leukemia.

The malignant cells in a patient with hairy cell leukemia responded most evidently to lipopolysaccharide (LPS) in in vitro culture for 3 1/2 days when the conventional tritiated thymidine uptake method was used. Since the malignant cells from patients with several other forms of leukemia and the peripheral blood mononuclear cells from healthy individuals did not show a comparable degree of responsiveness to LPS, we could exclude the possibility that this response was due to effects on contaminating normal mononuclear cells or to the nonspecific conditioning effect through LPS-affected contaminating normal monocytes. Morphological changes were observed with photo- and electronmicroscopy. It is likely that the hairy cells from the patient did respond to LPS, and whether or not this phenomenon may be confined to this type of lymphoid leukemia is not being investigated.     

Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells

The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T- cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.     

Non-leukemic dividing cells in the blood of leukemic patients.

Spontaneous mitoses in the blood of 67 patients with acute leukemia were enumerated and their identity determined by cytogenetic methods. Most patients were children with acute lymphoblastic leukemia. Simultaneous 16- to 20-hour cultures of blood leukocytes (Bu) and of bone marrow (BM) cells were performed without phytohemagglutinin (PHA). Blood leukocytes were also cultured with PHA for 72 hours (BPHA). Mitoses in Bu cultures were counted, and karyotypic analysis performed on cells from the three culture types. In 21 control subjects, Bu cultures usually yielded no mitoses. Relapse and remission patients both displayed significantly more Bu mitoses than the controls. The karyotypes of Bu, BPHA, and BM mitoses in remission patients were normal. Fifty percent of relapse patients displayed cytogenetically abnormal leukemia cell lines; the percentage of their abnormal karyotypes was significantly higher in BM cells than in Bu or BPHA cells. The majority of the mitotic cells in Bu cultures from both relapse and remission patients appear to be of a nonleukemic origin. The number of mitoses could not be correlated with type of leukemia, hematologic parameters, or prognosis.     

Surface immunoglobulins on hairy cells of 55 patients with hairy cell leukemia

Surface immunoglobulins (SIg) were determined on peripheral blood samples from 55 patients with hairy cell leukemia (HCL) and on hairy cells from spleen preparations of 14 of these 55 patients. The patterns of SIg for HCL was compared to the patterns on peripheral blood leukemic cells from 39 patients with chronic lymphocytic leukemia (CLL) and 15 patients with poorly differentiated lymphocytic (PDL) lymphoma. Of the 55 HCL patients, 42 could be scored for individual heavy and light chains; 16 had only IgG, 14 had two or three heavy chains, 7 had only IgD, and 5 cases had no SIg and were E-rosette negative. This pattern was different from the B-cell pattern in CLL and PDL where there were few cases of IgG alone (5%) and many cases of IgM alone (50%). Surface marker profile did not correlate with survival in any of the sub-groups tested. HCL appears to be a B-cell lymphoproliferative disease in greater than 90% of cases; many combinations of heavy chains with only a single light chain can be demonstrated.     

Alterations in the phenotype of hairy cells during culture in the presence of PHA: requirement for T cells

Culture studies of peripheral blood mononuclear cells from 7 entirely typical cases of hairy cell leukemia showed that after culture in the presence of PHA for 2--5 days, the predominant cell type changed from E- SIg+ CIg+ gamma FcR+ muFcR+ hairy cells to an E+ SIg- CIg- gamma FcR- muFcR- population of transformed cells derived from hairy cells. Depletion and readdition experiments demonstrated that cell-to-cell contact with T cells was necessary for the phenotypic change, while several observations indicated that the E+ population was not derived from T cells present before culture. The E positivity of the cultured cells was shown to be due to the possession of E receptor not acquired from the culture fluid, but the cells differed from true T cells in lacking both mature and immature T-cell antigens. The relevance of these in vitro observations to the continuing controversy concerning the nature of the hairy cell and to the in vivo fluctuations in immunologic phenotype not infrequently observed in hairy cell leukemia is briefly discussed.     

Hairy cell leukemia and other "hairy-cell" lymphoproliferative disorders (LPD-HC): the significance of morphologic, histologic and immunologic studies

In this report the clinical, morphologic, histologic and immunologic findings of 41 patients with hairy lymphoid cells in peripheral blood and/or bone marrow are analyzed. In 27 patients the diagnosis of hairy cell leukemia was established. 14 patients had other variants of lymphoproliferative disorders: malignant lymphoma with hairy cells--7, chronic lymphocytic leukemia with hairy cells--5, and T cell lymphoproliferative disorder with hairy cells--2 patients, respectively. Several variants of malignant lymphoma with hairy cells were defined: lymphocytic, centrocytic and lymphoplasmacytic. The importance of combined use of bone marrow biopsy and immunophenotyping for the correct diagnosis of hairy cell leukemia and other "hairy-cell" lymphoproliferative disorders is stressed. The obtained data suggest relationship between characteristic clinical manifestation (isolated splenomegaly), presence of hairy cells and CD11c-antigen expression.     

Surface immunoglobulin density on human peripheral blood mononuclear cells.

The densities of surface immunoglobulin (slg) on peripheral blood mononuclear cells (PBM) of normals and patients with chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (LCL), and hairy cell leukemia (HCL) were analyzed using the fluorescence-activated cell sorter (FACS). PBM were labeled with fluorescein conjugates of F(ab')2 fragments of affinity chromatography-purified anti-Fab or class-specific anti-mu, anti-delta, anti-gamma, or anti-alpha. Histograms of relative cell fluorescence, rems of relative cell fluorescence, reflecting slg density, were prepared with the FACS. Anti-Fab-labeled normal PBM demonstrated a homogeneous low-density peak that when separated by the FACS and analyzed cytochemically consisted predominantly of monocytes, whereas brighter-staining cells were predominantly lymphocytes. Anti-mu and anti-delta labeled 9.0% and 8.5% of normal PBM, respectively, the slg+ cells being virtually all lymphocytes. Cells labeled by anti-gamma exhibited low homogeneous slg density and consisted of more than 80% monocytes. No normal or leukemic PBM were labeled by anti-alpha. All slg-positive cells (less than 5% monocytes) from 12 of 13 patients with CLL had very low homogeneous densities of slg and bore slgM, Whereas cells from 9 of 13 and 2 of 13 patients bore slgD and slgG, respectively. Similarly, PBM from 2 patients with HCL exhibited low and homogeneous densities of algM, slgD, and slgG, whereas those from a third patient bore only slgG. By contrast, the density of slgM and PBM derived from 3 patients with LCL was very high; slgD and slgG densities varied from very high to undetectable in these patients. The different homogeneous densities of slg on peripheral blood lymphocytes from patients with CLL, HCL, and LCL suggest that these diseases represent malignant transformation of different B-lymphocyte subpopulations.