Interleukin-4 in the treatment of AIDS-related Kaposi’s sarcoma

Annals of Oncology 8: 79-83,1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Original article Interleukin-4 in the treatment of AIDS-related Kaposi's sarcoma A. Tulpule,1 B. Joshi,2 N. DeGuzman,1 B. M. Espina,1 R. Mocharnuk,1 O. Prakash,2 D. Templeton,3 A. M. Levine1 &P. S.Gill1 1

Department ofInternal Medicine, Division of Hematology, University of Southern California School of Medicine, Los Angeles, CA; 2Alton Ochsner Medical Foundation, Division of Research, New Orleans, LA, USA; 3'University ofGlasgow, Scotland, UK

Purpose: To define the safety and toxicity of interleukin-4 (IL-4) when administered subcutaneously in patients with AIDSrelated Kaposi's sarcoma (AIDS-KS); to evaluate the effect of IL-4 on immunologic and virologic parameters; and to preliminarily assess the response rate of IL-4 in AIDS-KS. Patients and methods: Eighteen patients with mucocutaneous, non-visceral AIDS-KS were treated with IL-4 at a dose of 1 mcg/kg subcutaneously, daily until unacceptable toxicity or for a maximum period of six months. Twelve (66%) patients had extensive mucocutaneous disease with over 25 lesions. Ten patients had received prior systemic chemotherapy. Seventeen had CD4+ lymphocyte counts less than 200/mm3. Results: The most common adverse effects included headache in 78%, fever in 56%, chills in 44%, and edema in 44%. Hematologic toxicities consisted of grade 4 neutropenia (less than 500/mm3) in 33%, mild anemia in 22%. Transient elevation

Introduction Kaposi's sarcoma (KS) is the most common neoplasm associated with human immunodeficiency virus (HIV) infection and approximately 10% of all cases with Acquired Immunodeficiency Syndrome (AIDS) first present with KS [1]. KS is a multifocal disease with skin involvement as the most common site, but progression to the lymph nodes, mucus membranes, lungs and gastro-intestinal tract is common. Patients with early disease and CD4 lymphocyte counts over 200/mm2 have been shown to respond well to interferon-a [2, 3]. Other modalities such as cytotoxic chemotherapy have been very effective in treating patients with more advanced and aggressive disease and less intact immunity [4, 5]. However, the majority of patients relapse after cessation of therapy. Thus, new therapies which can be delivered safely over long periods are needed. Interleukin-4 (IL-4) was initially described as a regulator of growth and differentiation of B-cells [6]. IL-4 has a broad spectrum of cell activating properties that are not confined to the B-lymphocyte system. IL-4 is capable of increasing viability and stimulating growth in normal helper and suppressor T-cells [7] as well as other T-cell lines [8, 9] and synergzes with IL-7 in the production of

of liver enzymes was noted in 17%. A transient elevation in CD4+ lymphocyte counts occurred during the first two weeks of therapy. Four of eleven patients tested showed marked decline in plasma HIV RNA after four weeks. Partial remission was observed in one patient, lasting six months. Three other patients (17%) had stable disease: 7 weeks in one patient, and 10 weeks in each of the two other patients. Conclusion: Grade 4 neutropenia (absolute neutrophil count < 500/mm3) was the most common hematologic adverse effect with IL-4 in patients with AIDS-KS. In contrast to in vitro findings, there was a decrease in plasma HIV RNA after four weeks of IL-4 therapy in the majority of patients tested. IL-4 produced minimal anti-tumor effects in AIDS-KS with one partial remission in a patient with CD4 lymphocyte counts over 200/mm3. Further studies of IL-4 in AIDS-KS may be considered in patients with better immune status. Key words: AIDS-related Kaposi's sarcoma, interleukin-4

lymphokine-activated killer cells (S. Rosenberg, personal communication). IL-4 has been shown in vitro to inhibit the growth of a variety of tumor types including plasmacytoma, mammary adenocarcinoma, melanoma and sarcoma cell lines [10]. IL-4 also inhibits the growth of AIDS-KS derived spindle cell lines in vitro [11]. IL-4 has been tested in patients with various malignancies [12-17], One complete remission has been observed in a patient with advanced refractory Hodgkin's disease [18]. We describe the results of an open-label phase II trial designed to determine the safety and toxicity of IL-4 when administered subcutaneously in patients with AIDS-related Kaposi's sarcoma. The secondary objective was to evaluate the immunologic, virologic and antitumor effects.

Patients and methods Patients HIV seropositive adult patients with biopsy proven Kaposi's Sarcoma (KS) were accrued between October 1992 and May 1994. Eligible patients had 10 or more mucocutaneous KS lesions without symptomatic visceral involvement and a WHO performance status of 0, 1 or 2. Required laboratory criteria included a WBC count greater than

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Summary

80 2,500/mm3 or granulocyte count greater than 1,500/mm3, platelet count greater than 75,000/mm3, serum bilirubin less than 2.0 mg/dl, liver enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase) less than three times normal, creatinine level less than 2.0 mg/dl and creatinine phosphokinase (CPK) and lactate dehydrogenase (LDH) within normal limits. Exclusion criteria included use of steroids within 14 days of study entry; biologic response modifiers, or systemic chemotherapy within three weeks of study entry; radiation therapy or investigational therapy within four weeks of study entry. Patients with symptomatic AIDS-defining opportunistic infections or with other acute intercurrent infections within two weeks were not eligible. Other exclusion criteria included history of cardiac dysfunction or peptic ulcer disease and upper gastrointestinal bleeding.

Patient evaluation Prior to therapy, the number of cutaneous lesions were counted and characterized as raised or flat in each patient. Five indicator lesions were selected, measured bi-dimensionally and assessed monthly. In patients with less than 50 total skin and oral lesions, all lesions were evaluated for changes in the number of lesions and characteristics. In patients with greater than 50 lesions, a representative area was chosen for tumor evaluation. Patients were initially evaluated with complete blood counts, serum chemistries, peripheral blood CD4 and CD8 lymphocyte counts, p24 antigen and [32-microglobunn; in patients with elevated lactate dehydrogenase or creatinine phosphokinase levels, the cardiac isoenzyme fractions had to be within normal limits. Chest radiograph, electrocardiogram and MUGA scan for left ventricular ejection fraction were also performed prior to study entry. Hematology and blood chemistries were repeated weekly for four weeks, then biweekly thereafter. Patients were restaged monthly for response to therapy and monitored biweekly for toxicities.

Treatment regimen Patients were treated with interleukin-4 (IL-4, Schering-Plough Corporation, Kenilworth, NJ) at a dose of 1 meg/kg/day subcutaneously and were observed for any adverse effects for at least two hours following injection of IL-4 for the first two days of therapy. After two treatment days with observation, patients were taught how to self-administer IL-4 for subsequent doses. Patients were treated for six months or until another study endpoint: documented disease progression, intolerable adverse effects as denned by an unresolved or recurrent grade 3 toxicity, or any grade 4 toxicity using the Southwest Oncology Group toxicity grading criteria. Patients were required to be on a stable antiretroviral dosing schedule or regimen with a licensed agent(s) (zddovudine, didanosine, zalcitabine) for at least one month prior to study initiation and to remain on that same regimen for the duration of the study except in the case of unacceptable toxicity attributable to the antiretroviral agent(s).

Response evaluation Complete remission (CR) was defined as the absence of any detectable residual disease, including tumor associated edema, persisting for at least four weeks with biopsy confirmation of absence of KS from one representative residual pigmented cutaneous lesion. Partial remission (PR) was denned as complete flattening of at least 50% of all previously

Virologic evaluation Quantitative competition (QQ polymerase chain reaction (PCR) for measurement of HIV RNA species was performed to measure viral load from plasma samples stored at -80 °C using, methods previously described [19].

Results Sixteen homosexual and two heterosexual males with nonvisceral AIDS-KS were accrued. The median age was 36 years (range 25-59). Ten patients were Caucasian and eight Latino. Baseline demographics are provided in Table 1. Nine patients had received prior systemic chemotherapy with a combination of doxorubicin (adriamycin), bleomycin, and vincristine (ABV); one other patient received multiple regimens with vincristine, vinblastine, etoposide and doxorubicin. Three patients had received prior therapy with interferon-oc. Toxicities Headache was the most common non-hematologic toxicity, experienced by 14 (78%) (Table 3). Mild to moderate fever occurred in 10 (56%), and chills and edema in eight (44%) patients each. Less common adverse effects included anorexia, cough, and proteinuria (two each, 11%). Anxiety, appetite changes, generalized weakness, pruritus, sleep changes, tingling and gastritis were seen in one patient each. Hematologic adverse effects were dose limiting in six patients. Grade 4 neutropenia (absolute neutrophil count less than 500/mm 2 ) was observed in six (33%) patients. The median time to onset of grade 4 neutropenia in these six patients was 19 days from the initiation of therapy. Table I. Disease characteristics. Median time from first KS Range (months) KS distribution > 50 lesions 25-50 lesions < 25 lesions B-symptoms Prior systemic chemotherapy Prior local therapy Radiation Intralesional Antiretroviral therapy AZT alone or combination ddl or ddC alone CD4 lymphocytes < 200/mm3

18 months 3-57 8 (44%) 4 (22%) 6 (33%) 6 (33%) 10(56%) 5 (28%) 2(11%) 12(67%) 6 (33%) 17(94%)

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The initial five patients enrolled on the study all had poor immune function and advanced disease and were studied for HIV burden. Serum p24 antigen, plasma and peripheral blood mononuclear cells (PBMC) culture, CD4 and CD8 lymphocyte counts, and (32-microglobulin were performed to ensure that there was no significant HIV stimulation by IL-4. The study was suspended at that point for analysis of the above parameters. After no upregulation of HFV was observed, additional patients were accrued. In addition to the above studies, at the completion of the trial, HIV RNA was quantitatively measured from stored plasma collected at baseline and week 4.

existing lesions for at least four weeks; or a 50% decrease in the sum of the products of the largest perpendicular diameters of the marker lesions without development of new lesions or worsening of tumor associated edema. Progressive disease (PD) was defined as a 25% increase m the number, size or character (from flat to raised) of preexisting lesions or new visceral sites of involvement Stable disease (SD) was defined as any response not meeting the criteria for CR, PR or PD.

81 Table 2 Entry diagnostic and median laboratory parameters.

LVEF% CD4 lymphocytes (/mm3) CD8 lymphocytes (/mm 3 ) p2-microglobulins AGC (/mm3) Hemoglobin (gm/d) Platelets (xlOOO/mm3) HIV RNA (copies/ml)'

a)

Median

Range

60% 12 401 i,S 2258 12.5 187 280,000

51%-76% 1-335 76-1250 2.5-5.8 1006-12,367 8.4-16.5 91-337 8,500-3,681,480

§

100 E

a

__—-—'

S

— ,

10

o

1

Abbreviations: LVEF - left ventricular ejection fraction; AGC - absolute granulocyte count

The median absolute neutrophil count (ANQ nadir was b) 436 (range 313-490). All patients with an ANC of less than 500 were removed from the study and treated with granulocyte colony stimulating factor (G-CSF). No patients developed neutropenic sepsis or complications 1000 from neutropenia. Of the patients who developed anemia, none had a hemoglobin of less than 8.0 g/dl. Four patients had a hemoglobin between 8.0 and 10.0 g/dl, and 100 of these, the median nadir was 8.9 g/dl and median time to onset was 13 days (range 7 to 17 days). There were no instances of thrombocytopenia less than 75,000/mm3. Transient elevations of liver enzymes (ALT and AST) occurred in three patients. There were no instances of hyperbilinibinemia or elevations in the cardiac isoenzyme fractions in LDH or CPK. Figure 1

Effect of IL-4 therapy on peripheral blood CD4+ (a) and CD8+ (b) lymphocyte counts. The changes observed at weeks 2 and 4 were not statistically significant compared to basehne.

Immunologic and virologic evaluations CD4+ and CD8+ lymphocyte counts during the first month of therapy are summarized in Figures la and lb. A transient elevation in CD4+ lymphocytes was observed. However, no statistically significant changes were observed in CD4+ or CD8+ lymphocyte counts (P > 0.05

Table 3. Non-hematologic, most severe reported toxicities (n - 18). Total

Grade

Headache Fevers Chills Edema Night Sweats Vomiting Myalgia Arthralgia Fatigue Tachycardia Diarrhea Flu-symptoms Malaise Nausea Pain Rhinitis

Weeks on Therapy

1

2

3

4

n

11 5 7 5 4 5 3 3 3 4 3 3 2 3 2 3

2 3 1 1 1 0 0 1 1 0 0 0 1 0 1 0

0 2 0 1 0 0 1 0 0 0 0 0 0 0 0 0

1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

14(78%) 10(56%) 8 (44%) 8 (44%) 5 (28%) 5 (28%) 4 (22%) 4 (22%) 4 (22%) 4 (22%) 3(17%) 3(17%) 3(17%) 3(17%) 3(17%) 3(17%)

by analysis of variance) or (32-microglobulin (data not shown) over time. Two patients were p24 antigen positive at study entry. Of these two, one became p24 antigen negative after two weeks of therapy and this was persistent to week 20 of therapy. The other patient did not undergo repeat p24 antigen testing. AJ1 other patients initially p24 antigen negative remained negative throughout the study. Repeat plasma specimens on eleven patients were evaluated for HIV RNA by RT-PCR from stored plasma at study entry and after 28 days of therapy (Figure 2). Four patients showed one log or greater decline compared to baseline in RNA copies/ml of p24 antigen by RT-PCR after four weeks of therapy. Six patients showed less than one log decline in RNA copies of p24 antigen compared to baseline by RT-PCR. Only one patient had an increase of less than one log compared to baseline in RNA copies of p24 antigen. This patient came off IL-4 after three weeks of therapy due to grade 4 neutropenia and the plasma was collected after the last dose of IL-4. Therapy summary and response to therapy Seven (39%) patients came off study because of unacceptable toxicity: six patients developed neutropenia less than 500/mm3, and one developed grade 4 edema (anasarca).

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Weeks on Therapy

10,000,000 *

1,000,000 -

If.

'a. o u

100,000

•<

X

u 0-

Pre

Week 4

Figure 2. Measurement of plasma HIV-RNA by RT-PCR during IL-4 therapy. Eleven patients were studied at baseline and week 4 on therapy. More than a log decline in plasma HIV-RNA was noted in 4 and less than one log decline in six.

One other patient was taken off protocol for non-compliance with study visits. The median duration of therapy was four weeks (range 1-24 weeks). Major response was observed in one patient who demonstrated a partial remission, lasting six months. This partial responder had previously achieved partial remission with prior systemic chemotherapy and then relapsed before entering this trial. Nine patients have come off therapy for progressive disease after a periods of 3-10 weeks. Three of nine patients have had stable disease for 7,10, and 10 weeks.

Discussion The predominant clinical side effects observed in our study were headache, fever, chills and edema. These side effects are comparable to those seen in phase I trials of IL-4 [12-14], although headache was more common in our study (78%). The risk of headache in our patients may be increased secondary to HIV infection and elevated endogenous levels of cytokines. In contrast to other IL-4 studies, grade 3 elevation of liver enzymes (greater than five times the upper limit of normal) was relatively uncommon. Elevation in liver enzymes occurred in only two patients (11%), and resolved spontaneously in both cases. The suggestion of Gilleece et al. that IL-4 causes transient hepatic damage [12], particularly in patients with pre-existing liver disease, was not supported in our study. Of the four patients with

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Z OS

prior history of viral hepatitis, only one had elevation of liver enzymes during IL-4 therapy. Hyperbilirubinemia and significant elevations of alkaline phosphatase were not observed. IL-4 enhances HIV-1 replication in vitro [20]. Our study was the first to examine in vivo effects of IL-4 on HIV replication. The study was thus designed to accrue five patients to evaluate HIV replication and show that it is safe before further accrual. Eligible patients for this trial were therefore required to be on a stable antiretroviral therapy prior to and for the duration of the study. None of the five patients had increase in viral replication and thus the study was completed. Contrary to in vitro findings, we instead observed marked inhibition of viral replication in 4 of 11 patients studied, and a modest decrease in another six. Thus, in vivo effects do not parallel in vitro effects. These findings are not surprising based on the effect of IL-4 on various pro-inflammatory cytokines including TNF, IL-1, and IL-6 which are all known to enhance viral replication [21, 22] while IL-4 inhibits expression of these cytokines [23, 24]. These findings also argue against the belief that elevated expression of IL-4 in peripheral blood mononuclear cells of patients with HIV infection is detrimental to the long term outcome of disease [25]. It is apparent that the effects of IL-4, like other cytokines, are much more complex. IL-4 is known to increase viability and cell replication in normal CD4+ ancd CD8+ T-cells [7] and T-cell lines [8, 9]. In our study there was a transient increase in CD4+ lymphocyte count, but the CD8+ lymphocytes showed a decrease during the study that was not statistically significant. Human IL-4 has been shown to interact with hematopoietic growth factors and enhance production of IL-3 or GM-CSF and replication of multipotential, megakarocytes, myeloid, and erythroid cell lineages. Previous clinical trials of IL-4 support these observations. For example, a moderate but significant increase in neutrophils was noted by Gilleece et al. [12] while elevation of neutrophil count was noted in 3/19 patients by Prendiville et al. [13]. In contrast, we observed grade 4 neutropenia in six patients (33%). Neutropenia in our trial may be a result of IL-4 alone or the combined effects of IL-4 and antiretroviral agents. The patient who developed partial remission to IL-4 was the only patient who had a baseline CD4+ lymphocyte count over 200/mm3. Of note, this patient had received prior systemic chemotherapy which therefore did not appear to affect the ability to respond. However, it is likely that, as with interferon-a, a stronger predictor of response to IL-4 therapy may be patients with an intact immune systems. Therefore additional studies with IL-4 in patients with higher CD4+ lymphocyte counts are warranted.

83 Acknowledgement We are indebted to our patients for their continued participation in these studies, and to the excellent nursing and support staffs at the 5P21 AIDS clinic. Supported in part by a grant from Schering-Plough Corporation.

15.

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References

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Received 2 September 1996; accepted 3 December 1996.

Correspondence to: Anil Tulpule, MD Parkash S. Gill, MD University of Southern California Norris Cancer Hospital and Research Institute 1441 Eastlake Avenue, MS 34 Los Angeles, CA 90033 USA

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