Loss of interferon antibodies during prolonged continuous interferon-
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Loss of interferon antibodies during prolonged continuous interferon-
From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 1991 77: 792-798 Loss of interferon antibodies during prolonged continuous interferonalpha 2a therapy in hairy cell leukemia RG Steis, JW 2d Smith, WJ Urba, DJ Venzon, DL Longo, R Barney, LM Evans, LM Itri and CH Ewel Updated information and services can be found at: http://www.bloodjournal.org/content/77/4/792.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on November 7, 2014. For personal use only. Loss of Interferon Antibodies During Prolonged Continuous Interferon-a2a Therapy in Hairy Cell Leukemia By Ronald G. Steis, John W. Smith II, Walter J. Urba, David J. Venzon, Dan L. Longo, Ruth Barney, Lydia M. Evans, Loretta M. Itri. and Cynthia H. Ewe1 Although highly active in hairy cell leukemia (HCL), interferons (IFN) are not curative in this disease; current data indicate that prolonged IFN therapy will be necessary t o control disease in the majority of patients. We previously observed acquired IFN resistance in association with neutralizing IFN-a2a antibodies in small numbers of patients with HCL. This finding suggests that the requisite long-term therapy may be compromised if there is an increasing incidence over time of neutralizing antibodies. We performed a follow-up study of IFN antibodies in our patients receiving continuous IFN therapy. All 16 patients who were previously antibody negative remained so. Surprisingly, all nine patients who previously had non-neutralizing IFN antibodies became antibody negative after a median of 14.5 months. Moreover, 3 of 10 patients who had neutralizing antibodies became antibody negative and five had only non-neutralizing antibod- ies a median of 10 months from the time neutralizing antibody had first been detected. Only two patients had persisting neutralizing antibodies. Inhibition of neopterin synthesis, inhibition of generation of 2‘, 5’ oligoadenylate synthetase activity, and inability t o detect IFN in serum after subcutaneous injection of IFN-a2a was observed only in the one patient tested with neutralizing IFN antibodies confirming that these antibodies have functional significance in vivo. We conclude that, although neutralizing IFN antibodies inhibit the effectiveness of IFN in vivo, these antibodies are produced only transiently during long-term therapy. The long-term effectiveness of this drug will not likely be affected in most patients by neutralizing antibody. This is a US government work. There are no restrictions on its use. I if not all, patients will require repetitive courses of therapy or chronic maintenance therapy to maintain normal peripheral blood (PB) counts. We have been studying the long-term effectiveness of IFN in 56 patients with HCL. In contrast to other studies in which there was a predetermined duration of IFN administration and in light of inevitable disease progression after cessation of therapy, we opted to administer IFN continuously until either disease progression or unacceptable toxic effects developed. The results of this study will be published separately.8During the course of this study, we observed a 31% incidence of IFN-neutralizing antibody formation and the development of acquired IFN resistance in approximately one-third of the patients who had neutralizing antibodies? The antibodies were specific for IFN-a2a; there was no cross-reactivity with partially purified natural IFN. Resistance to the antitumor effects of IFN was not observed in the absence of neutralizing antibodies. Because prolonged use of IFN will be necessary for the long-term management of this disease, the development of neutralizing IFN antibodies and IFN resistance is a potentially significant problem that may affect an increasing proportion of patients over time. To further study this issue and to study possible alternative therapies for patients who develop antibody-related IFN resistance, we determined the effect of IFN antibodies on IFN-induced changes in serum neopterin levels and PB mononuclear cell Z‘, 5‘ oligoadenylate synthetase activity. Such analyses were performed following doses of either IFN-a2a (the IFN species these patients had been receiving) or human lymphoblastoid IFN to investigate the specificity of neutralizing antibodies in vivo. We also reevaluated serum specimens from our patients late in the course of IFN therapy for the presence of IFN antibodies. NTERFERON a (IFNa) has been used as an effective agent in the treatment of hairy cell leukemia (HCL) since its activity in this disease was established by Quesada et a1 in 1983.’ A number of have shown that the majority of patients will have a clinically meaningful improvement in peripheral blood counts with IFN doses that have minimal toxic effects. This therapy has become the treatment of choice for HCL patients who progress after splenectomy and is indicated for that subset of newly diagnosed patients who are unlikely to benefit from splenectomy.’ Studies are ongoing to determine if IFN should replace splenectomy as initial therapy for other patient subsets.‘ Despite this impressive activity in HCL, IFN is not curative in this disease. Only rarely do patients have well-documented complete remissions and disease progression is the rule following even up to 18 months of continuous therapy.’ The preponderance of data suggest that most, From the Biological Response Modifiers Program, Clinical Research Branch, and Program Resources, Inc, National Cancer InstituteFrederick Cancer Research and Development Center (NCI-FCRDC), Frederick; Frederick Memorial Hospital, Frederick; Biostatistic and Data Management Section, Clinical Oncology Program, Bethesda, MD; and Clinical Investigation, Hofmann-LaRoche, Inc, Nutley, NJ. Submitted June 11, 1990; accepted October 17, 1990. Supported in part with Federalfunds from the Department of Health and Human Services under contract number NOl-CO-74102. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S.Government. Address reprint requests to Ronald G . Steis, MD, NCI-FCRDC (BRMP), FMH Cancer Treatment Center, Clinical Research Branch, 502 W Seventh St, Suite No. 3, Frederick, MD 21 701. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. This is a US government work. There are no restrictions on its use. 0006-4971I9117704-0008$0.0010 792 PATIENTS AND METHODS Patients. Before receiving IFN therapy, the diagnosis of HCL was established in all patients using previously reported criteria.“ All patients were required to have one or more PB cytopenias Blood, Vol77, No 4 (February 15). 1991: pp 792-798 From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 793 LOSS OF INTERFERON ANTIBODIES (hemoglobin less than 12 g/dL, platelets less than lW,WO/mm3,or granulocytes less than 1,500/mm3). All patients were treated according to the same protocol as outlined previo~sly.~~'~ Each initially received 3 million units of recombinant I F N - d a (rIFN-u2a) (Roferon-A; Hoffmann-LaRoche, Nutley, NJ) daily by subcutaneous injection for approximately 6 months. The dose in responding patients was then reduced to 3 million units administered thrice weekly. Maintenance therapy at this dosage was continued indefinitely as long as responses were maintained with acceptable side effects. Several patients who did not respond initially or who stopped responding were given higher doses of IFN for variable periods of time. The in vivo effects of IFN-a2a antibodies were determined by serially measuring serum levels of IFN and neopterin and PB mononuclear cell-associated 2', 5' oligoadenylate synthetase (2-5 A) activity in patients following a single dose of either IFN-cr2a or human lymphoblastoid IFN. Patients who were still receiving maintenance therapy discontinued IFN treatments 2 weeks before this evaluation. Baseline blood samples were obtained three times for measurement of these parameters. Patients then received 9 million units of I F N - d a subcutaneously. Serial blood samples were obtained for IFN levels at 0, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 hours after the IFN injection and for neopterin levels and 2-5 A activity at 0, 4, 8, 24, and 48 hours after IFN injection. One week later, baseline blood samples were obtained again and patients were then administered 9 million units of human lymphoblastoid IFN (WELLFERON, Burroughs Wellcome Research, Triangle Park, NC) subcutaneously. Serial blood samples were again obtained as previously described for measurement of the same parameters. Patients were not given acetaminophen for fever but were offered meperidine for chills following the administration of IFN. Vital signs were obtained serially before and after IFN administration. Both studies were approved by the Investigational Review Boards of the Frederick Cancer Research and Development Center and the Clinical Oncology Program of the National Cancer Institute. All patients gave written informed consent. IFN antibody assays. Serum samples were screened for the presence of antibodies against IFN-a2a by a solid-phase enzyme immunoassay (EIA) as described by Itri et al." Specimens testing positive in the EIA were assessed for their ability to neutralize the antiviral effects of rIFN-a2a or natural IFN-a in vitro in an antibody neutralization bioassay (ANB)."Neutralizing antibodies were considered to be present if the specimen neutralized the viral protective effects of IFN in bovine kidney cells exposed to vesicular stomatitis virus (VSV). Results are expressed as neutralization titers, ie, that dilution of serum that reduced the effective concentration of IFN from approximately 20 U/mL to 1 U/mL. The antibody titer, expressed as IFN-neutralizing U/mL (INU/mL), is equivalent to the number of units of IFN neutralized by 1mL of the patient's serum; under the assay conditions described, it is defined as the neutralization titer times 20. Serum IFN levels. IFN levels were determined by inhibition of the cytopathic effect of VSV on the human amnion WISH cell line (ATCC, Rockville, MD) as described previously.'* Serial threefold dilutions of serum samples were added to WISH cell monolayers and incubated overnight at 37°C. VSV was added and cell viability was graded from 0 (no kill) to 4 (total kill) after 24 hours. The endpoint IFN titer was defined as the last serum dilution to show 50% protection of the monolayer. IFN units were defined as 3N where 3 is the base dilution and N equals the endpoint IFN titer. Neopterin determinations. Neopterin levels, which are expected to increase following the administration of IFN," were measured by a commercial radioimmunoassay (Neopterin-RIAcid, Henning, Berlin, Germany). Briefly, sera were incubated with an anti- neopterin antibody followed by addition of an 'ZSI-neopterintracer. Serum neopterin concentrations were calculated from the percent drop in 1Z51-binding when compared with control. 2'-5'-oligoadenylate synthetase (2-5 A ) assay. 2'-5'-oligoadenylate synthetase activity was measured as described by Merritt et al.14Ficoll-hypaque separated PB mononuclear cells were lysed and incubated with poly rI.rC agarose beads to allow adherence of the 2-5 A enzyme. Beads were then incubated for 20 hours with 'H-adenosine triphosphate (ATP) buffer solution followed by digestion with bacterial alkaline phosphatase. Five-microliter aliquots were spotted onto diethyl aminoethyl (DEAE)-cellulose paper disks, washed, eluted with 0.3 mol/L KCI, and counted. Specific activity was defined as picomoles ATP incorporated per 10s cells per hour. One unit of enzyme activity was defined as the incorporation of 1pmol/L ATP/hour at 37°C. RESULTS IFN antibodies. Between April 1984 and December 1985, 56 patients with HCL were entered on study and began treatments with IFN-a2a. Of these, 35 subsequently were studied to determine the in vivo effects of IFN in the presence or absence of IFN antibodies. Thirty-two of these 35 patients were receiving IFN up to 2 weeks before study entry while three patients had discontinued IFN 6, 15, and 17 months before study entry. IFN was stopped in these three patients because of acquired IFN resistance (one patient) or because of what were felt to be IFN-related side effects (two patients). IFN antibody analyses of these 35 patients (Table 1) showed that many patients who previously had IFN antibodies became antibody negative during continuing IFN administration. For example, of the 10 patients who previously had neutralizing antibody, three became antibody negative, five continued to have antibody that no longer had neutralizing activity, and only two continued to have neutralizing antibody. One of these latter two patients had developed IFN resistance when neutralizing antibodies developed' and discontinued IFN 17 months before participation in this study. However, at the time he entered this study he had become neutralizing-antibody negative. The loss of IFN antibodies observed in our study population despite continued exposure to IFN prompted us to evaluate the antibody status of the 21 remaining patients who had not entered this second study. Serum specimens were available from 16 of these patients. Of three patients who previously had non-neutralizing IFN antibodies, one Table 1. Antibody Status of Patients Receiving Continuous IFN-a2a Therapy for HCL ~ No. PatientstPrior Antibody Status Current Antibody status* Median (range) Duration of Antibody Positivity (mo) 16 Antibody negative 9 Non-neutralizing 10 Neutralizing 16 Antibody negative - ~ 9 Antibody Negative 3 Antibody negative 5 Non-neutralizing 2 Neutralizing 14.5 (2-48) 10 (2-34) *"Current antibody status" refers to the patients' antibody status at the time of entry onto the study or, if IFN had been stopped, at the time IFN was discontinued. From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 794 had become antibody negative and two continued to have non-neutralizing antibodies. Eight patients previously had neutralizing antibodies; two became antibody negative, two continued to have antibody but without IFN-neutralizing activity and four continued to have neutralizing antibodies. All five antibody-negative patients remained antibody negative. Thus, in total, all 21 patients who were antibody negative previously remained antibody negative, 10 of 12 patients with non-neutralizing antibodies became antibody negative, and 12 of 18 patients with neutralizing antibodies became either antibody negative (five patients) or continued to have antibody that no longer had neutralizing activity (seven patients). Of the remaining six patients with persisting neutralizing IFN antibodies, five had either de novo (one patient) or acquired (four patients) IFN resistance and remained neutralizing-antibody positive at least until IFN therapy was discontinued. One patient with persisting neutralizing antibody continues on IFN therapy. The median duration of antibody positivity among the 35 patients who entered our study with neutralizing or nonneutralizing antibodies was 10 months (range 2 to 34 months) and 14.5 months (range 2 to 48 months), respectively. The two patients who continued to have neutralizing antibodies have had them for 7 and 35 months. Those five patients who converted from neutralizing to non-neutralizing antibodies had neutralizing antibodies for a median of 14 months (range 2 to 18 months). The median duration of IFN therapy among those patients who never developed detectable IFN antibodies was 46 months. The specificity of the antibody response to IFN was tested in vitro in a viral protection assay using either IFN-a2a or partially purified natural IFN. All neutralizing IFN antibodies detected in our patients were specific for IFN-a2a. No inhibition of the viral protective effects of partially purified natural IFN was observed. In vivo effects of IFN antibodies. Subcutaneous administration of both IFN-a2a and human lymphoblastoid IFN brought about consistent and reproducible increases in serum levels of neopterin, IFN, and PB mononuclear cell-associated levels of 2-5 A in patients without antibodies and in those with non-neutralizing antibodies (Fig 1). Non-neutralizing IFN antibodies had no statistically significant effect (by the Wilcoxon rank sum test) on the increase over baseline of neopterin or 2-5 A levels when compared with what was observed in these same patients following human lymphoblastoid IFN administration or compared with the antibody-negative group following IFN-a2a administration. Serum IFN levels increased to a significantly greater degree following human lymphoblastoid IFN than following IFN-a2a administration in both the antibodynegative and non-neutralizing antibody groups. In the one patient with neutralizing IFN antibodies, serum IFN was not detectable and there was no or very little increase in neopterin and 2-5 A levels following subcutaneous injection of IFN-a2a. However, following subcutaneous injection of the same number of units of human lymphoblastoid IFN, changes in IFN, neopterin, and 2-5 A levels were similar to STEIS ET AL Neutralizing Ab Positive = -2. E 1 400 - 300 - -Interferon : b = IFNp2a 500 200- -50 -40 -30 -0- Neopterln -20 ..e..2 ’ 5 ’ - A c 100r 4 - 2 0 2 4 6 8 -+ -.E i 30 -25 5 .- -20 5 0 -15 f 0 -10 5 ul -10 -5 0 -0 N 10 Non-Neutralizing Ab Positive IFN,uZa I 1 4 - 2 0 2 6 4 IO 8 Antibody Negative IFNunl IFNa2a 100 I I 160 -2. E 30 E e c :. 40 b -c c 20 10 -2 2 4 6 8 1 0 Day Relative to Start of Therapy Fig 1. Changes in IFN, neopterin, and 2-5 A levels in a representative patient from each group following subcutaneous administration of either IFN-da or human lymphoblastoid IFN. Nine million units of IFN-u2a was administered on day 0 and the same amount of human lymphoblastoid IFN was administered on day 7. those observed in patients with either no antibody or only non-neutralizing antibody. Body temperature changes were monitored for 12 hours following each IFN injection (Fig 2). Given the limited number of patients who continued to have neutralizing IFN antibodies, statistical comparisons of the febrile response to the IFNs is not possible. However, the one patient with neutralizing IFN-a2a antibodies had only a transient temperature increase following IFN-a2a injection that was of shorter duration than patients with either no antibody or only non-neutralizing antibodies. However, this same patient had as high and as prolonged a temperature increase following human lymphoblastoid IFN injection as the other patients. Although patients with non-neutralizing antibod- From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 795 LOSS OF INTERFERON ANTIBODIES 40 - "'1 0 Ab Positive Ab Positive Neutralizing Non-Neu t ralizing 3gl Ab Negative 3g,61 I p n=' 39 '( n-6 39 II 39 n924 3 4 ' 36" o i ' z ' s ' 4 ' ~ . ' ' ' 8 7 e ' . ' 9 ' m 36,6 SI ' n w L o i a 3 4 Hours 6 e. 7 a o i o n w 36.6 9 .1 . . ' ' ' o i z ' s Hours ' 4 ' 6 ' ' 8 7 ' a ' o ' ' m n u ' Hours Fig 2. Body temperature changesfollowing subcutaneous administrationof 9 million units of either IFN-a2a (H)or human lymphoblastoid IFN (0). Curves show median temperatures for each patient group. Standard deviations for each point were usually less than 0.2% and are not included in the graphs for clarity. The discrepancy in the number of patients with non-neutralizingantibodies between this figure and Table 1 is because one patient had neutralizingantibody when IFN-&a was discontinued but had a non-neutralizing antibody when he entered our second study. Four patients in the antibody-negative group were excluded from this analysis because two did not complete the study and two inadvertentlyreceived acetaminophen. ies showed a trend toward lower temperatures following IFN-a2a than after human lymphoblastoid IFN, the difference was not statistically significant. Relationship of IFN antibody status and clinical outcome. The presence of neutralizing IFN antibodies has previously been correlated with acquired resistance to the antileukemic effects of IFN in patients with HCL.' Despite substantial inhibition of IFN-induced changes in body temperature and in 2-5 A and neopterin levels, our one remaining patient with IFN-neutralizing antibodies has continued to maintain a partial response to IFN therapy with sustained improvements in PB counts and bone marrow (BM) hairy cell infiltration. However, he is the only patient receiving continuous IFN-a2a therapy who has a progressively increasing soluble interleukin-2 (IL-2) receptor level (data not shown). Levels of the soluble IL-2 receptor have previously been correlated with disease status in HCL.15316 The increasing soluble IL-2 receptor level in this patient with neutralizing IFN antibodies suggests that disease progression may be occurring despite continued IFN-a2a administration. The clinical relevance of loss of neutralizing IFN antibodies is illustrated in the clinical course of the patient shown in Fig 3. This patient developed resistance to IFN at approximately day 100 of therapy. At about the same time, the IFN dose was reduced from 3 million units daily to 3 million units thrice weekly. Decreasing granulocyte and platelet counts did not improve when IFN was again administered at 3 million units daily but blood count improvement was observed following dose escalation to 6 million units daily. However, when she began treatments at this dose neutralizing antibodies were no longer detectable. Subsequent IFN-dose reductions to 3 million units daily and 3 million units thrice weekly, which previously were ineffective in the presence of antibody, were now effective at maintaining normal PB counts in the absence of antibody. Thus, antibody-mediated IFN resistance was overcome by increasing the IFN dose and the effective dose of IFN subsequently decreased to the "standard" dose level (3 million units thrice weekly) when neutralizing antibodies no longer were detectable. DISCUSSION The use of IFNa in the treatment of malignant and infectious diseases is increasing. Although currently approved for use in HCL and in acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma, recombinant forms of IFNa are also significantly active in indolent non-Hodgkin's lymphoma^,'^ chronic myelogenous leukemia (CML),18.'9 and chronic active hepatitis.'" One study has also suggested that IFNa in combination with 5-fluorouracil is highly active in previously untreated patients with colorectal carcinoma." In all of these diseases, complete remissions are uncommon and, where evaluated, disease progression following cessation of therapy is the r ~ l e . ~ ,The *~,~ development of IFN-neutralizing antibodies, observed with both commercially available forms of IFNa, and the correlation of the presence of these antibodies with acquired disease resistance to IFNy,,Z*1,Z5 raises the possibility that the prolonged IFN therapy anticipated for these diseases might not be possible. Increasing proportions of patients might be expected to develop antibodies to IFN during long-term therapy, rendering IFN ineffective. A greater understanding of the effects of these antibodies on easily measurable in vivo parameters of IFN activity might allow rational therapeutic alterations in patients with antibody-mediated IFN From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 796 STEIS ET AL IFN Dose EIA ANB -I 3 MUqd : .3MUqd+3MUtiw + + + + : + + - 6 MUqd 3 MUqd-3 t - I 4 MUtiw- (150)/600)(1200)/1200)(1200]i3001 I II III I I 6,000 600 5,000 ,500 4,000 ,400 % 3,000 300 4 I 8 . (* V 1 c E LI : 1 1 1 8 3 2 200 2,000 -4 0 1,000 loo 51 9 2 $ 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 Day of Therapy resistance. Therefore, we studied the in vivo effects of IFN antibodies in a population of patients with HCL, some of whom had developed antibodies to IFN-a2a, to determine the in vivo effects of these antibodies and to determine if serologically distinct IFN species could overcome antibodyrelated resistance to IFN-a2a. We first determined the incidence of IFN antibodies. To our surprise, the majority of patients who were previously antibody positive had become antibody negative despite continuous exposure to IFN-a2a. The loss of serum IFN antibodies was seen in patients with a wide range of titers (300 to 6400 INU/mL), not only in those with the lowest values. The assay used in this study had a lower limit of sensitivity of 100 INU/mL and the lowest neutralizing antibody titer previously observed in our patients was 300 INU/mL. Furthermore, all patients in our study had discontinued IFN treatments 2 weeks before study entry. Loss of IFN antibodies was, therefore, not due to recent IFN administration, which can acutely lower antibody titers." Alterations in the immunogenicity of the IFN-a2a administered to our patients also does not explain loss of IFN antibodies because the processes used in IFN-a2a manufacture have not changed (Itri, L., personal communication, March 1990). Thus, either immunologic tolerance to IFNa2a developed in these patients or the IFN antibodies were produced by short-lived B cells without the development of immunologic memory. Loss of IFN antibodies has clinical relevance in some patients. The acquired IFN resistance of the patient described in Fig 3 may have been overcome by an increase in IFN dose. But an increase in IFN dose to 6 million units daily was made at the same time that neutralizing IFN antibodies became undetectable. Thus, responsiveness to IFN may have been restored because of loss of IFN antibodies rather than to the higher IFN dose. Certainly, 6 Fig 3. Serial granulocyte and platelet counts in a patient receiving continuous IFN-a2a for HCL. IFN doses and results of both EIA and ANB for IFN antibodies are shown across the top. Numbers across the top refer to neutralizing antibody titers. the continued response to IFN-a2a at a dose of 3 million units three times weekly must have been due to loss of IFN antibodies because disease progression had occurred at this IFN dose when neutralizing antibodies were present. Therefore, if patients develop IFN antibodies and IFN resistance, higher IFN doses may overcome the resistance but may be required for only a short period of time (in our patient, less than 10 months). Serial measurements of IFN antibodies may be helpful in guiding the management of such patients. Resistance to IFN is not universal among patients with HCL who develop neutralizing IFN antibodies. In one study, only 6 of 16 patients with neutralizing IFN antibodies experienced disease progression despite continued IFN administration.' This observation may be explained by the indolent nature of this disease. In the Cancer and Acute Leukemia Group B study of IFN-a2b in HCL: disease progression requiring reinstitution of therapy occurred a median of approximately 2 years following cessation of either 12 or 18 months of therapy. Therefore, in patients initially responsive to IFN who develop antibody-mediated IFN resistance, a median of as long as 2 years might elapse before IFN resistance becomes evident clinically, even among patients with antibody titers sufficiently high to neutralize all IFN administered. The results shown in Fig 1 are consistent with this interpretation in that acute changes in levels of neopterin and 2-5 A induced by IFN were nearly completely blocked by neutralizing IFN antibodies. The antibodies were sufficiently potent to completely block the appearance of IFN-a2a in the serum following subcutaneous administration. Despite these inhibitory effects of the antibody, this patient is maintaining a partial response to IFN therapy. That these inhibitory effects are due to antibody and not to a generalized unresponsiveness to IFN is confirmed by the normal expected response of neopterin and 2-5 A levels to human lymphoblastoid IFN, a prepara- From www.bloodjournal.org by guest on November 7, 2014. For personal use only. 797 LOSS OF INTERFERON ANTIBODIES tion of IFN that contains many different molecular species of IFNa, most of which are serologically distinct from IFN-a2a. These results show that IFN-neutralizing antibodies inhibit IFN responses in vivo. However, the clinical effects of this inhibition appear to depend upon the natural history of the disease being treated. The indolent nature of HCL may permit interference with the antileukemic effects of IFN to occur for a prolonged period of time without clinical deleterious effects. It is of interest that the development of IFN-a2b neutralizing antibodies in patients with CML, a disease generally more rapidly progressive than HCL, is nearly universally associated with clinical disease progressionF6 Alterations in the management of patients with disease responsive to IFN is not required solely because of the development of neutralizing IFN antibodies. Antibodymediated resistance to IFN may be overcome by increasing the IFN dose. Even if higher doses are ineffective, patients who can safely sustain loss of IFN effectiveness for several months may ultimately lose neutralizing IFN antibodies with return of IFN effectiveness. Animal studies have shown that antigens to which tolerance can develop are, in general, slowly metabolized, presumably resulting in prolonged in vivo exposure.’’ If tolerance induction is the mechanism by which patients lose IFN antibodies, continuous IFN therapy may be required to induce tolerance and in this regard may be preferred over intermittent therapy, at least in patients with HCL. Monitoring of antibody levels should continue to be a routine part of the clinical evaluation of IFN and other recombinant biologic agents. REFERENCES 1. Quesada JR, Reuben J, Manning JT, Hersh EM, Gutterman JU: Alpha interferon for induction of remission in hairy-cell leukemia. N Engl J Med 310:15,1984 2. Quesada JR, Hersh EM, Manning J, Reuben J, Keating M, Schnipper E, Itri L, Gutterman JU: Treatment of hairy cell leukemia with recombinant a-interferon. Blood 68:493,1986 3. 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