Pulse Interleukin-2 with Famotidine Induces CD56+ Lymphocytes in the Peripheral Blood of Patients with Metastatic Melanoma or Kidney Cancer

Abstract

Increased lymphokine-activated killer (LAK) cell numbers and cytotoxicity against tumor cell lines have been seen in patients receiving high-dose continuous and bolus infusion interleukin-2 (IL-2) regimens. LAK are CD56 positive on flow cytometry. Daily intravenous doses of IL-2 of 18–21.6 MIU/m² over 15–30 minutes (“pulses”) have been developed to attempt to lessen the toxicity of this therapy. It has been previously shown that the patients with metastatic melanoma or kidney cancer may be treated safely with pulse IL-2 daily for 5 days preceded by intravenous famotidine. Cycles were repeated every 21 days. Because LAK numbers have not been previously described with this regimen, the present study has examined CD56 numbers via peripheral blood flow cytometry in 11 patients with samples scheduled at baseline, after two cycles, and after four cycles. Eight (8) patients had melanoma and 3 had kidney cancer. Median CD56 counts after two cycles was significantly higher than baseline (p = 0.001). Similarly, CD56 counts at 2 months later were also greater than baseline (p = 0.009). There was no difference between median values after two cycles versus after four cycles. Patients who were clinical responders had a median CD56 count of 650 after two cycles when compared with nonresponders who had a median CD56 count of 290 (p = 0.005). CD56 counts are significantly elevated in patients treated with pulse IL-2 with famotidine and clinical responders have significantly higher CD56 than nonresponders.

Introduction

Interleukin-2 (IL-2)–activated lymphocytes may mediate the destruction of a variety of tumor lines.¹ When exposed to high IL-2 doses, CD56-positive activated lymphocytes are detected by flow cytometry and are called lymphokine-activated killer (LAK) cells.^2

–5 IL-2 as a single agent is able to mediate durable antitumor responses in patients with metastatic melanoma and kidney cancer, although considerable toxicity due to increased capillary permeability may be seen.⁶

Famotidine has been associated with increased tumor cell cytotoxicity by lymphocytes in vitro.⁷ This is postulated to be due to increased internalization of IL-2 by IL-2 receptors on T lymphocytes and natural killer cells. The serum famotidine level needed for this effect corresponds to what is achievable clinically with intravenous administration.

Daily intravenous doses of IL-2 of 18–21.6 MIU/m² over 15–30 minutes (“pulses”) have been developed to attempt to lessen the toxicity of IL-2 therapy. The combination of pulse IL-2 with famotidine is currently being tested in patients with metastatic melanoma and kidney cancer.^8
–10 The present study was done to examine the effect of pulse IL-2 in terms of LAK generation in vivo.

Patients and Methods

The present study's pulse IL-2 regimen with famotidine has been previously described in detail.^8
–10 Briefly, patients were treated with a dose of IL-2 of 18–21.6 MIU/m² in 50 mL of 5% dextrose intravenously over 15–30 minutes for 5 consecutive days. All patients received famotidine 20 mg intravenously preceding the daily IL-2 dose. Cycles were repeated every 3 weeks for four cycles, then every 3–4 weeks for two cycles, and every 4–6 weeks in the absence of disease progression or intolerable toxicity.

All patients had measurable evidence of histologically confirmed metastatic melanoma or kidney cancer. They were required to have Eastern Cooperative Oncology Group performance status <1, or an estimated survival of at least 3 months, white blood cell count >3500 per mm³, platelets >100,000 per mm³, hemoglobin >9.0 g/dL, total bilirubin, ALT, and AST <3 × upper limit of normal, and serum creatinine <2.0 mg/dL. Patients were excluded if they had autoimmune diseases such as inflammatory arthritis, which could potentially be exacerbated by IL-2; any medical illness requiring corticosteroids or other immunosuppressive agents such as methotrexate; current untreated brain metastases; or a history of significant cardiovascular disease including myocardial infarction, congestive heart failure, primary cardiac arrhythmias, angina pectoris, or cerebrovascular accident. Informed consent was obtained from all patients prior to the study.

Prior to treatment on this regimen, all patients had to have undergone low-level cardiac stress test and/or cardiac evaluation to exclude occult atherosclerotic heart disease.

Eleven (11) patients were enrolled on this study. Peripheral blood flow cytometry was done on samples scheduled at baseline, after two cycles, and after four cycles. Venous blood was collected into ethylenediaminetetraacetic acid and lithium heparin vacutainer tubes (Becton-Dickinson and Company, Franklin Lakes, NJ). Lymphocyte subsets were analyzed using either an EPICS XL or FC500 flow cytometer and the automated TetraONE system (Beckman Coulter, Hialeah, FL). Cell staining was performed either manually or on a Prep PlusII automated processor using 100 μL of heparinized whole blood with 10 μL of Cyto-stat TETRA chrome CD45-FITC/CD4-RDI/CD8-ECD/CD3-PC5 or Cyto-stat TETRA chrome CD45-FITC/CD56-RDI/CD19-ECD/CD3-PC5 antibodies, followed by red cell lysis using TQ PREP (Beckman Coulter). The samples were analyzed using TETRA chrome software (Beckman Coulter), which gates on CD45 bright, low side scatter lymphocyte population for subset percentage determination. Daily instrument settings were optimized with Flow-check, Flow-set beads, and Cyto-trol or fresh normal donor stained control cells (Beckman Coulter). Two levels of Immunotrol Control Cells (Beckman Coulter) were confirmed within tolerance limits prior to each patient run. Absolute subset count enumeration was calculated from white blood cell counts and differential obtained using ethylenediaminetetraacetic acid–whole blood on LH-750 cell counter (Beckman Coulter). Subset lymphocyte counts and percentages were reported for CD3−/CD56+ cells. Student's t-test was used to compare values for the groups at the different intervals.

Results

Five (5) patients with melanoma and 1 with kidney cancer have shown clinical response to therapy. Responding sites included lungs, liver, lymph nodes, and subcutaneous/soft tissue.

An increase in CD56-positive cells was noted in all treated patients. Median CD56 counts after two cycles was significantly higher than baseline (p = 0.001) (Table 1). Similarly, CD56 counts after four cycles were also greater than baseline (p = 0.009) (Table 1). There was no difference between median values after two cycles versus after four cycles. Patients who were clinical responders had a median CD56 count of 650 after two cycles when compared with nonresponders who had a median CD56 count of 290 (p = 0.005) (Table 2).

Table 1.

Comparison of Median CD56 Counts (mm ⁻ ³) at Baseline, After Two Cycles, and After Four Cycles

Baseline	After 2 cycles	After 4 cycles
130	430	338

p = 0.001 for after two cycles (range: 160–710) versus baseline (range: 80–260). p = 0.009 for after four cycles (range: 210–443) versus baseline. p-value is not significant for after two cycles versus after four cycles.

Table 2.

Comparison of Median CD56 Counts (mm ⁻³) After Two Cycles in Clinical Responders Versus Nonresponders After Two Cycles

Responders	Nonresponders
650	290
(range: 360–710)	(range: 160–460)

p = 0.005.

Discussion

CD56, previously known as Leu19, is a 220,000 molecular-weight protein present on the majority of lymphocytes that exhibit nonmajor histocompatibility complex-restricted cytotoxicity against tumor cells.² IL-2, administered in high-dose bolus or continuous infusion schedules, increases the number of CD56-positive lymphocytes in peripheral blood.^2,3,5

Two groups have identified CD56+ cells in patients receiving daily intravenous IL-2 doses on schedules different from the one in the present study. Mitchell et al. treated patients with 10 daily doses of 21.6 MIU/m² and a single infusion of cyclophosphamide over the course of a 3-week cycle.^11,12 Hersh et al. used intravenous doses of IL-2 up to 36 MIU/m² on Monday, Wednesday, and Friday each week.¹³

No prior studies have been published describing the effect of short daily infusions of IL-2 and famotidine on the number of CD56-positive cell numbers in the blood. In the present study, CD56-positive cells were elevated after treatment with pulse IL-2. These cells persisted after additional cycles. The presence of CD56+ cells correlated with clinical response.

Previously, Mitchell et al. reported increased “LAK-like” cytolytic activity in patients with melanoma who responded to a daily IL-2 dose of 21.6 MIU/m².¹¹ LAK cytotoxicity was not measured in the present study, but will be the subject of a future trial.

The presence of CD56+ cells in peripheral blood does not necessarily implicate these lymphocytes as the effectors of antitumor response. Their appearance could simply indicate that the subject's immune function is sufficient for such an event. Nevertheless, if similar results are corroborated by larger studies using pulse IL-2 regimens with famotidine, CD56 levels may be useful as a prognostic indicator for the patients with metastatic melanoma or kidney cancer.

Conclusions

Daily pulse IL-2 preceded by famotidine results in elevated CD56 counts. CD56 counts are significantly elevated in clinical responders compared with nonresponding patients.

Footnotes

Acknowledgments

The authors gratefully acknowledge the support by the Brody Medical Foundation and the Owen Hendrixson Memorial Fund.

Disclosure Statement

No completing financial interests exist.

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