Alpha-interferon and lymphokine-activated killer cells in hairy cell leukemia

Lymphokine-activated killer (LAK) cell activity generated from peripheral blood was tested in 6 patients with typical hairy cell leukemia, 3 not on treatment with alpha-interferon (alpha-IFN) and 3 receiving therapy. In all cases, substantial killing of the LAK-sensitive target Daudi was observed, but hairy cells, whether or not they had been pretreated with alpha-IFN, were uniformly resistant to LAK lysis. The hairy cells were also resistant to LAK cell killing generated from normal peripheral blood mononuclear cells. alpha-IFN added at various times during LAK generation had little or no effect on LAK activity. It is concluded that LAK cells are not important in mediating the beneficial effects of alpha-IFN in hairy cell leukemia.     

Interleukin 12 Augments Natural Killer-Cell Mediated Cytotoxicity in Hairy Cell Leukemia

Interleukin 12 (IL12, NKSF-natural killer stimulatory factor) was found to stimulate natural killer (NK) cell activity of hairy cell leukemia (HCL) patients. Two patients not responding to IL12 stimulation were also resistant to interferon alpha (IFN alpha)-mediated augmentation of NK activity. IL12 also enhanced slightly the interleukin 2 (IL2)-induced cytotoxicity of HCL patients, while IFN alpha reduced the stimulatory effect of IL2. These data suggest that interleukin 12 has NK modulatory properties in HCL leukemia patients, which may be different from those of IFN alpha.     

Whole blood assay for NK activity in splenectomized and non-splenectomized hairy cell leukemia patients during IFN-alpha-2b treatment

Natural killer cell (NK) activity in peripheral blood (PB) was followed longitudinally for up to 2 yr after initiation of low-dose IFN-alpha-2b therapy in nine hairy cell leukemia (HCL) patients. A whole blood NK (WB-NK) assay was employed in order to measure the NK activity per unit blood. The pretreatment WB-NK activity was consistently low, indicating that the patients' total NK activity in PB is decreased. Striking differences in WB-NK activity were observed between splenectomized and non-splenectomized patients, whereas no consistent patterns were found when using the conventional NK assay. Thus the WB-NK activity of splenectomized patients showed an immediate increase after initiation of treatment, while the activity in non-splenectomized patients decreased and remained low during the first 3-6 months. Subsequently, after reduction in spleen size, the WB-NK activity began to increase. In splenectomized patients, a second rise in WB-NK was observed after 3-6 months of therapy, coinciding with the normalisation of the peripheral blood counts. In both groups of patients incubation with IFN in vitro induced a rise in NK activity before start of treatment, which was abrogated promptly after initiation of therapy, indicating a maximal in vivo boosting of the NK cells. These differences observed indicate that the response of splenectomized and non-splenectomized HCL patients to IFN treatment should be evaluated separately.     

Cytotoxic in vitro function in patients with metastatic renal cell carcinoma before and after alpha-2b-interferon therapy. Effects of activation with recombinant interleukin-2.

In this study the characteristics of the cytotoxic function in a series of patients with metastatic renal cell carcinoma (RCC) were analyzed and the possibility of modulating this capacity in vitro with the use of biologic response modifiers (BMR) such as alpha-interferon (alpha-IFN) and recombinant interleukin-2 (rIL-2) was verified, with the ultimate goal of providing a rationale for a therapeutic approach to this disease with these molecules. Peripheral blood mononuclear cells (PBMC) of patients with advanced RCC were tested for natural killer (NK) and lymphokine-activated killer (LAK) activity both before and after alpha-IFN therapy. In addition, surface markers of unstimulated and stimulated cells were analyzed and in vitro assays were performed to determine the proliferative capacity in response to the stimulus with rIL-2. During an evaluation before treatment, defective NK activity was observed that could be corrected by incubating the cells with rIL-2. In these subjects, LAK cells could be consistently generated after PBMC were activated with this cytokine in vitro. No changes in NK and LAK activity were found after alpha-IFN therapy. In contrast, treatment with alpha-IFN affected the proliferative response of PBMC to rIL-2, and a significant decrease in this in vitro capacity was observed during follow-up. The ability to restore NK activity and obtain an adequate LAK cytotoxicity from the PBMC of patients with RCC supports a therapeutic approach with BRM. However, the fact that this type of treatment affects the proliferative response of PBMC to rIL-2 must be considered when clinical trials are designed for patients with RCC.     

Development of lymphoproliferative disorder of granular lymphocytes in association with hairy cell leukemia

Lymphoproliferative disorder of granular lymphocytes (LDGL) is a low grade T-cell disease characterized by clonal expansion of large granular lymphocytes of either T cell or natural killer (NK) cell lineage that express the cytotoxic T-cell/NK cell antigens CD16, CD56 and/or CD57. LDGL has been described in association with other malignancies, leading to theories of a common abnormal stem cell as well as development of the LDGL as an immune response to a primary tumor. We have studied 32 patients with hairy cell leukemia (HCL). In 15 patients (47%) we detected an increase in cells expressing cytotoxic T-cell/NK cell antigens. In 10(31%) patients these cells were of T cell lineage, while 5 patients (16%) had increased NK-cells. T cell clonality was detected by PCR in all cases with increased cytotoxic T-cells in which adequate DNA was obtained from peripheral blood. Since in 2 patients the LDGL was not present at diagnosis but developed during follow up, our data suggests that clonal LDGL may develop in response to the HCL. The significance of LDGL in the setting of HCL and flow cytometric evaluation of HCL versus LDGL will be discussed.     

Is there a direct differentiation-inducing effect of human recombinant interferon on hairy cell leukemia in vitro?

We used a panel of monoclonal antibodies (moAb) to label splenic hairy cells from eight patients to determine the membrane phenotypes, the presence of cytoplasmic immunoglobulin (cIg), and the expression of maturation-associated antigens. All eight patients had responded clinically to splenectomy either alone or in combination with alpha-2b-interferon (alpha-IFN) therapy. For each sample, cytofluorimetric analysis showed distinct, and in six cases multiple, heavy chain isotypes. After short-term culture in the presence of alpha-IFN or gamma-interferon (gamma-IFN), samples from four patients displayed characteristic changes in surface immunoglobulin (sIg) expression. When compared with untreated cells, cells co-cultured with alpha-IFN or gamma-IFN showed in four and three patients, respectively, changes that were consistent with a shift to the more mature stage in B-cell ontogeny. However, in parallel with the changes in the sIg isotypes, treatment with IFN did not induce the appearance of cIg nor did the staining patterns for moAb to CD5, CD19, CD20, and CD22 antigens indicate the induction of terminal maturation. These data suggest that hairy cell leukemia (HCL), a neoplasm of "mature" B-cells, is potentially susceptible to maturation stimuli. Based on these findings, it might be of interest to examine whether co-factors, which have proved to play a role in HCL (e.g., B-cell growth factor [BCGF]), are capable of further enhancing IFN-induced differentiation.     

Reduced hematologic response to alpha-interferon therapy in patients with hairy cell leukemia showing a peculiar immunologic phenotype.

Alpha-interferon (alpha-IFN) treatment is highly effective in normalizing the clinical, hematologic, and immunologic parameters of patients with hairy cell leukemia (HCL). Complete remissions (CR), however, are rare, and a few patients do not respond adequately to alpha-IFN. That the poor response to alpha-IFN treatment could be related to a particular immunologic surface marker profile of the HC was investigated in this study. The results showed that most patients who do not respond adequately to alpha-IFN HC have a peculiar immunologic phenotype with a positive response to the Leu1 (CD5) monoclonal antibody, usually absent on HC but characteristically expressed on B-chronic lymphocytic leukemia cells. Of nine HCL patients with this phenotype, only three had partial remissions (PR) and six minor responses (MR) compared with the three CR, 16 PR, and three MR observed in the 22 Leu1 (CD5)-negative patients. The authors postulate that a more extensive immunologic analysis of HCL patients at diagnosis may be predictive of the response to IFN treatment.     

Effect of alpha-IFN on cytokine-induced antigen expression and secretion of TNF, LT and IgM in HCL

In order to investigate the possible mechanisms for the effect of alpha-interferon (alpha-IFN) in hairy cell leukaemia (HCL), blood cells from 4 cases were treated in vitro with alpha-IFN, tumour necrosis factor (TNF) and interleukin 2 (IL-2). Changes in the antigen expression, immunoglobulin (Ig) secretion and the production of TNF and lymphotoxin (LT) were investigated. TNF induced expression of CD4 and CD71, increased the intensity of HLA-DR, CD25, CD11c and CD13 expression and decreased both the intensity and frequency of sIg and cIg positivity. alpha-IFN decreased CD25 expression, the tartrate-resistant acid phosphatase activity (TRAP), reduced the TNF-induced CD4 and CD71 expression and antagonized the TNF effect on the Ig expression. Spontaneous TNF or LT production could not be detected in culture supernatants. However, TNF was found to induce LT production, an effect which alpha-IFN antagonized and IL-2 augmented. The reduction of CD25, TNF-induced CD71 and TRAP caused by alpha-IFN seems to represent a deactivation of the activated state of hairy cells (HCs). The failure of alpha-IFN to induce Ig secretion or CD38 expression in HCs speaks against a differentiation induction effect. The LT secretion induced by TNF suggests that other cytokines than TNF might be involved in the proliferation of HCs and that alpha-IFN by blocking the production of LT and perhaps other cytokines causes a growth arrest in HCs.     

Effect of alpha-interferon on the immune system of patients with hairy cell leukemia

The role of in vivo administration of lymphoblastoid alpha-interferon (IFN-alpha) on the immune status of 12 patients with hairy cell leukemia (HCL) was studied. In most cases an increase in T3(CD3), T4(CD4), and T8(CD8) lymphocyte subsets was documented at the same time as hairy cells disappeared from the circulation. This led in most cases to an improved T4/T8 ratio. The effect of treatment with IFN-alpha was particularly evident on the natural killer cell compartment, which is often functionally depressed in HCL at diagnosis. After therapy, a progressive increase in the cytotoxic activity was observed in most patients. This was more evident 6-9 months after commencing treatment. These findings suggest that, in addition to the known clinicohematological effects, IFN-alpha can improve both the T and natural killer compartments in patients with HCL.     

Serum soluble interleukin-2 receptor levels in hairy cell leukemia: correlation with clinical and hematological parameters and with alpha-interferon treatment.

The soluble Interleukin-2 Receptor (sIL-2R) serum levels were assessed in 42 patients with Hairy-Cell Leukemia (HCL) at diagnosis and after alpha-Interferon therapy and correlated with spleen size, peripheral hematological values, hairy cell index (HCI) and clinical response. Serum sIL-2R levels were significantly increased in all HCL patients, particularly in those with a higher HCI (> 0.50) and in non-splenectomized patients. Among the 26 HCL patients who were studied before and after 12 months of alpha-IFN treatment, 16 normalized the sIL-2R level and 10 did not. Our findings suggest that sIL-2R levels in HCL patients correlate with the splenic and bone marrow tumor burden as assessed by HCI. In addition patients with low levels of sIL-2R at diagnosis appear to have a better chance of achieving a good clinical response.     

Treatment of Hairy Cell Leukaemia with 2'-Deoxycoformycin

The adenosine deaminase inhibitor 2'-deoxycoformycin (DCF) has been used to treat 40 patients with hairy cell leukaemia (HCL) who have been followed up for a minimum of 6 months. 21 patients had previously undergone splenectomy. 33 had received treatment with alpha-interferon (IFN), half of whom had relapsed and the remainder had either been partially treated due to intolerance or had an incomplete response. Deoxycoformycin was administered by slow intravenous injection at a dose of 4 mg/m² weekly for 4 consecutive weeks and then 2-weekly for 4 doses followed by 4-weekly injections until a complete remission was achieved. No maintenance therapy was given. The overall response rate was 97yi, with 82% complete remission (CR) and 15% partial remission (PR). CR have lasted from 3+ to 30+ months (median 12 +) with no relapses recorded so far. Only one evaluable patient, suffering from a variant form of HCL, failed to respond to DCF, whilst two other HCL-variant cases who had been resistant to alpha-IFN have achieved a PR and are still on treatment. DCF was generally well tolerated, the major toxicity being related to cytopenia and associated infection. Four patients developed severe infections, one of whom died of septicaemia before it was possible to evaluate the response to DCF. A group of 12 patients who had received alpha-IFN immediately prior to treatment with DCF and had obtained clinical and haematological benefit had fewer infections with DCF than the group who had either not received IFN immediately before or who had failed to respond to it. In view of the infections associated with DCF, we would now recommend initial therapy for HCL patients with alpha-IFN for 2-4 months to obtain clinical and haematological improvement, followed by DCF given 2-weekly, to eradicate residual disease. This approach may achieve a higher proportion of sustained CR with a short treatment time and minimal toxicity.     

Decreased natural killer (NK) activity in patients with myeloproliferative disorders

Natural killer (NK) cell number and activity were measured in 26 patients with myeloproliferative disorders and the results were compared with 16 age-matched control patients. The percent of Leu-11b-positive cells was 11% +/- 3% in the patients, compared with 12% +/- 4% in the control patients. Ten of 26 patients, however, had NK activity lower than all of the control values at three different effector to target cell ratios (E:T) (P less than 0.005). The values of those patients with low unstimulated NK activity remained low despite stimulation with interleukin-2 (IL-2) or alpha-interferon (alpha-IFN), whereas the values of those patients with normal unstimulated activity responded to IL-2 and alpha-IFN like the control patients. Three of the ten patients with low NK activity had a history of malignant neoplasms. None of the 16 patients with normal NK activity had a history of malignant neoplasms (P less than 0.05). We conclude that patients with myeloproliferative disorders frequently have low endogenous NK cell activity in vitro. The dysfunction of the NK system appears to be intrinsic because the relative number of NK cells was similar to control values and the response to stimulation with IL-2 and alpha-IFN was suboptimal. There may be a relationship between low NK activity and the development of malignant disease in such patients.     

Interleukin 2- and Interferon Alpha Induced Natural Killer Cell Activity as a Marker of Progression in Hairy Cell Leukemia

Hairy cell leukemia (HCL), a rare B-cell chronic lymphoproliferative disorder, is often accompanied by immune abnormalities. A marked impairment of the natural killer cell-mediated cytotoxity (NK activity) has been reported in most patients at diagnosis. In the present report a long-term follow-up study of NK activity of splenectomized HCL patients is recorded. Among patients who persisted with stable disease two groups, one with normal NK activity, and another with low NK activity, could be recognized. Patients with progressive stage were characterized by a low NK cytotoxic activity. In vitro tests showed that interferon alpha (IFN-oc) and interleukin 2 (IL2) could increase the NK activity to normal levels only in HCL patients with stable disease, while in progressive HCL these cytokines showed a significantly decreased effect. These results indicate that cytokine-induced NK cytotoxicity appears to be a valuable parameter in assessing the stage of HCL.     

Studies on the optimal dose and the mode of action of alpha-interferon in the treatment of hairy cell leukemia

The therapeutic efficacy and side effects of alpha-2c-interferon (IFN-alpha 2c) treatment of hairy cell leukemia were compared between two different dose regimen: 10 patients received maximum tolerable doses of IFN-alpha 2c for 1 year (group A), and 11 patients received minimum doses of IFN-alpha 2c, which induced an optimal biological response (group B). Induction of neopterin excretion was chosen as the marker to define biological response and the dose of IFN-alpha 2c applied was on average only one tenth of that in group A cases. Average time of treatment in group B was 42 weeks. The data indicate that both dose levels are effective in the treatment of advanced hairy cell leukemia but that the low dose regimen is free of toxicity. Laboratory investigations on the mechanism of IFN-mediated remission in HCL further revealed that hairy cells are resistant to lysis by IFN-alpha activated large granular lymphocytes and that improved natural killer function subsequent to IFN-alpha treatment in vivo is primarily due to the disappearance of leukemic hairy cells which dilute the natural killer effector cells. These findings support the view of a direct antitumor activity of IFN-alpha as the main therapeutic principle.     

Evidence for an association between hairy cell leukemia and renal cell and colorectal carcinoma.

BACKGROUND. Hairy cell leukemia (HCL) has been associated with several disease states. In this study, a possible association is reported between HCL and renal cell carcinoma (RCC) and colorectal carcinoma (CRC). METHODS. A retrospective study of the case records of 50 patients with HCL in a study of alpha-interferon (alpha-IFN) treatment of HCL. RESULTS. Three of 50 patients with HCL studied had RCC, and 2 of these also had CRC. In addition, two other patients had CRC. The other malignant lesions developed either before or after the diagnosis of HCL. In all patients, the HCL responded to alpha-interferon (alpha-IFN), but in four patients, the second lesion was diagnosed during IFN treatment. CONCLUSIONS. These findings could indicate that IFN does not correct a possible common basic etiologic defect and shows that even early CRC and RCC do not respond to the IFN doses administered. These findings should be considered in future trials of IFN treatment of these diseases. The authors also recommend a reevaluation of the frequency of second malignant lesions in HCL; this may be important particularly with the increased survival in patients with HCL who receive alpha-IFN treatment.     

alpha-Interferon inhibits spontaneous and induced DNA synthesis in hairy cell leukemia.

The effect of alpha-interferon (alpha-IFN) on spontaneous or induced proliferation of isolated peripheral blood mononuclear cells from 5 hairy cell leukemia patients was studied. alpha-IFN inhibited the low spontaneous proliferation of B-ly7 positive hairy cells (HCs) and also the proliferation induced by tumour necrosis factor (TNF). Interleukin-2 did not affect HCs, but induced CD4 positive T cells to proliferate, an effect which alpha-IFN antagonized. The stimulatory effect of TNF on the growth of HCs proved to be reversible and was partially blocked with either anti-TNF receptor or anti-lymphotoxin antibodies. Cellular or secreted thymidine kinase levels reflected the proliferative state of HCs in response to different in vitro treatments, as confirmed by thymidine incorporation and cell cycle studies.     

Flow cytometry in hairy cell leukemia before and during interferon alfa-2b therapy

Mononuclear cells from 15 patients with hairy cell leukemia were studied before and during therapy with interferon alfa-2b (IFN) by regular peripheral blood differential counts and flow cytometry, using a panel of monoclonal antibodies (Moab). Seven leukemic phase patients (Group 1) had a mean leukocyte count of 48,870/microliter at entry with a mean absolute hairy cell (HC) count of 40,100/microliter. After 3 months on IFN, both parameters decreased significantly (WBC 3,500/microliter; HC count 130/microliter). In eight patients with a cytopenic form of the disease (Group 2) the mean leukocyte count rose from 2950/microliter to 3890/microliter while the mean absolute HC count decreased from 300/microliter to 120/microliter. The morphologic shifts correlated well with changes in the Moab reaction pattern. In Group 1 the activity of all Moab decreased significantly. In Group 2, only cells expressing Leu 3a and Leu 11a (a marker of natural killer cells) showed a significant shift, the latter increasing from 170/microliter to 360/microliter. This increase in natural killer cell antigen expression was not obvious based on routine morphologic observations alone. We show that flow cytometry may be a useful adjunct in monitoring the response of HCL to therapy. Changes in populations of cells that may be difficult to discriminate on morphologic grounds alone may be observed.     

Involvement of HLA Antigens, Interleukin 2 Receptor and B-Cell Growth Factor in Interferon Action in Hairy Cell Leukemia

Little is known about the mechanism(s) by which alpha-interferon (aIFN), when used as a biotherapeutic agent, suppresses the malignant cells and restores the normal phenotype of cells in patients with hairy cell leukemia (HCL). In previous studies using scanning electron microscopy (SEM) we found that alFN induced unique membrane alterations in target hairy cells in vitro. In addition, aIFN was shown to enhance the expression of HLA class II antigens on HCL cells, to induce the production of new proteins in such cells, and to lower the high levels of soluble IL-2 receptors in the serum of HCL patients. In the light of these results, and the fact that restoration of natural killer cell activity occurs in aIFN-treated patients well after the hematologic profile begins to improve, our studies have focused on the hypothesis that IFNs act directly on the target malignant cells, leading to the elimination of these cells either by inhibiting the proliferation of the malignant cells and/or triggering changes in the differentiation status of the malignant cells that lead to suppression (cytoconversion) of the malignant phenotype. We review the current hypotheses regarding alFN action on leukemic cells, with special reference to its potential antagonism with BCGF     

Prognostic variables in hairy cell leukemia after splenectomy as initial therapy

Splenectomy has been used as initial therapy in hairy cell leukemia (HCL) for many years and usually causes rapid improvement in peripheral blood counts. However, most patients eventually require further therapy. We have analyzed the case histories of 194 patients with HCL who had splenectomy as initial therapy. The median time to failure (second therapy or death) was 18.8 months. Univariate analysis of prognostic variables demonstrated that age, hemoglobin level, platelet count, bone marrow cellularity, bone marrow hairy cells, and hairy cell index (HCI) were statistically significant predictors of failure-free survival (FFS). However, only the bone marrow cellularity and platelet count were significant independent prognostic variables by Cox analysis. The patients were divided into the following three subsets by prognosis after splenectomy: (1) low risk of failure (cellularity less than 85% and platelet count greater than or equal to 60,000/microliters), (2) intermediate risk (cellularity less than 85% and platelet count less than 60,000/microliters), and (3) high risk (cellularity greater than or equal to 85%). The median FFS for each of these subsets was 56.5 months, 11.7 months, and 5.4 months, respectively. The median overall survival after splenectomy has not been reached with follow-up of up to 22 years. Patients diagnosed since 1982 have a better overall prognosis than those diagnosed earlier, with 4-year survivals of 88% and 68%, respectively (P less than 0.01). We conclude that splenectomy should continue to be the standard initial therapy in HCL. However, patients with bone marrow cellularity of 85% or greater have a short duration of response to splenectomy, and thus, may be considered for initial systemic therapy.     

Patterns of recurrence from head and neck cancer. An immunologic perspective

The influence of pretreatment natural killer (NK) cell activity on patterns of disease recurrence in patients with head and neck cancer was studied. One hundred eighty-three patients were followed prospectively for disease recurrence following definitive therapy (median disease-free follow-up = 15 months). Pretreatment peripheral blood NK cell activity against K562 target cells was measured for each patient and expressed in lytic units (LU). The risk of death caused by recurrent disease was greatest in those patients with peripheral blood NK cell activity less than 60 LU (P less than 0.05). The site of recurrence could also be distinguished by LU value. As a percentage of total recurrences, locally recurrent disease was predominant in the patients with NK function greater than 60 LU whereas distant metastases predominated in the group of patients with peripheral blood NK cell activity less than 60 LU. These patterns were maintained when patients were stratified by clinical N stage (N = 0 versus N+), T stage (T-1 or T-2 versus T-3 or T-4), and treatment regimen (surgery alone versus surgery and postoperative radiation therapy). Natural killer cell activity appears to have prognostic significance, both for disease-free survival and for site of recurrence independent of clinical stage. The indications for multimodality therapy for patients with head and neck cancer when stratified by NK cell function need to be clarified.