Immunomodulation with interferon-gamma and colony-stimulating factors for refractory fungal infections in patients with leukemia

Authors

  • M. Cecilia Dignani M.D.,

    1. Division of Supportive Care, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas
    Current affiliation:
    1. Infectious Diseases FUNDALEU (Foundation for the Fight Against Leukemia), Buenos Aires, Argentina
    Search for more papers by this author
  • John H. Rex M.D.,

    1. Centers for Infectious Diseases, University of Texas Medical School, Houston, Texas
    Current affiliation:
    1. AstraZeneca, Alderley Park, Macclesfield Chesire, SK10 4TF United Kingdom
    Search for more papers by this author
  • Ka-Wah Chan M.D.,

    1. Department of Pediatrics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
    Search for more papers by this author
  • Gordon Dow M.D.,

    1. Department of Infectious Diseases, The Moncton Hospital, Moncton, New Brunswick, Canada
    Search for more papers by this author
  • Margarida deMagalhaes-Silverman M.D.,

    1. Department of Internal Medicine, Division of Hematology and Oncology, School of Medicine, University of Iowa, Iowa City, Iowa
    Search for more papers by this author
  • AnneMarie Maddox M.D.,

    1. Division Hematology and Oncology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas
    Search for more papers by this author
  • Thomas Walsh M.D.,

    1. Immunocompromised Host Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
    Search for more papers by this author
  • Elias Anaissie M.D.

    Corresponding author
    1. Division of Supportive Care, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas
    • Division of Supportive Care, Myeloma Institute for Research and Therapy, Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, 4301 West Markham, Mail Slot 776, Little Rock, AR 72205
    Search for more papers by this author
    • Fax: (501) 686-6442


  • This article is a US Government work and, as such, is in the public domain in the United States of America.

Abstract

BACKGROUND

Invasive fungal infections (IFI) in immunocompromised patients are associated with significant morbidity and mortality, despite appropriate antifungal treatment and recovery from neutropenia. The outcome of these infections depends significantly on the overall state of immunosuppression, including mainly the phagocytic system (neutrophils and macrophages). Interferon-gamma (IFN-γ), granulocyte-colony—stimulating factor (G-CSF) and granulocyte-macrophage—colony-stimulating factor (GM-CSF) are cytokines that enhance the activity of neutrophils and macrophages.

METHODS

The authors reported 4 patients with leukemia and refractory invasive candidiasis or trichosporonosis despite 1–13 months of appropriate antifungal treatment.

RESULTS

Cytokines were administered for 1.5–5 months without significant toxicity. For each patient, initiation of interferon-gamma plus a colony-stimulating factor resulted in a clinical response. The contribution of cytokines to control the fungal infection in these 4 patients was suggested by the strong inflammatory reaction observed in the 2 patients who had an immediate response (within 7 days of initiation of cytokine therapy) and by the good outcome in the 2 other patients in whom antifungal agents were discontinued at the start of cytokine therapy.

CONCLUSIONS

These data suggested a potential role for immunomodulation in patients with leukemia with refractory invasive fungal infections. Cancer 2005. Published 2005 by the American Cancer Society.

Invasive fungal infections (IFI) in immunocompromised patients are associated with significant morbidity and mortality, even with the early institution of appropriate antifungal treatment and recovery from neutropenia. The outcome of these infections depends significantly on the overall state of immunosuppression.1

The phagocytic system (neutrophils and macrophages) constitutes an important defense against opportunistic fungal infection. Interferon-gamma (IFN-γ), granulocyte-colony—stimulating factor (G-CSF) and granulocyte-macrophage—colony-stimulating factor (GM-CSF) are cytokines that enhance the activity of neutrophils and macrophages and may, therefore, enhance the response to fungal infections.1

Since 1992, we began utilizing cytokines as salvage therapy for refractory IFI in patients with cancer with adequate neutrophil counts and remission of their underlying disease. Complete data were available for four patients. We report the response of these four patients to IFN-γ and a CSF.

MATERIALS AND METHODS

The clinical courses of the 4 patients with refractory IFI is summarized in Table 1. Patient 1 is a 14-year-old female with acute myeloid leukemia (AML) who developed chronic disseminated candidiasis (CDC) during disease remission induction chemotherapy with right upper quadrant pain, increased levels of serum alkaline phosphatase (AP) and bilirubin, and liver and spleen lesions as observed on computed tomographic (CT) scan images. After 5 months of antifungal treatment with amphotericin B (AMB) (total dose 3.4 g) and fluocytosine (5-FC), her condition improved and she received maintenance therapy with fluconazole. After receiving 1 month of fluconazole, she developed recurrent hepatosplenic lesions. Antifungal treatment was changed to AMB and then to amphotericin B lipid complex (ABLC) plus flucytosine (5-FC) and the patient underwent splenectomy and partial hepatectomy. Both tissue samples revealed fungal granulomas and yeast infections but cultures were negative. The clinical and radiologic findings resolved after 3 months of therapy (> 30 g total dose of ABLC) and 5-FC and fluconazole maintenance was resumed. Two months later, and while receiving fluconazole, the patient developed weight loss, increased serum levels of AP, and liver lesions as observed on CT scan images. She was considered refractory to antifungal agents (total of 11 months), fluconazole was discontinued, and cytokine therapy was initiated with 100 μg IFN-γ plus 250 μg/m2 GM-CSF 3 times a week. No other antifungal agent was added. A clear and rapid response ensued with normalization of AP serum levels and > 90% resolution of the hepatic lesions was observed on CT scan images obtained 30 days after initiating cytokine therapy. Complete resolution of liver lesions was documented 17 days after the end of cytokine therapy (EOT), which was received for a total of 2 months. The patient also regained her usual weight 3 months after EOT and was well and free of leukemia and candidiasis 3 years after EOT.

Table 1. Characteristics of Patients with Refractory Fungal Infections Treated with Cytokines
Patient no. Age (yrs)/genderMalignancy and treatmentFungal infectionAntifungal treatment received before cytokine therapyCytokines and changes in antifungal treatmentOutcome after cytokine therapy Day of first sign of response Last follow up
  1. wk: week; mos: months; 5-FC: 5-fluorocytosine; ABCD: amphotericin B colloidal dispersion; ABLC: amphotericin B lipid complex; ALL: acute lymphocytic leukemia; AMB: amphotericin B deoxycholate; AML: acute myeloid leukemia; BMT: bone marrow transplantation; EOT: end of treatment; G-CSF: granulocyte—-colony-stimulating factor; GM-CSF: granulocyte-macrophage—-colony-stimulating factor; GVHD: graft vs host disease; IFN-γ: interferon-gamma; L-AMB: liposomal amphotericin B.

Patient 1 14/femaleAML Induction chemotherapyChronic disseminated candidiasis11 mos AMB (3.4 g) + 5- FC (5 mo) Fluconazole (1 mo) ABLC (> 30 g) + 5-FC (3 mo) Fluconazole (2 mo)IFN-γ 100 μg + GM-CSF 250 μg/m2 3 × wk for 2 mo Fluconazole stoppedFavorable response Day 30 3 yrs after EOT: alive and well without leukemia or candidiasis.
Patient 2 22/maleALL Induction chemotherapyChronic disseminated Trichosporon beigelii infection3 mo AMB-ABLC (21 g) + fluconazole (3 mo) + 5-FC (34 days) AMB ocularIFN-γ 100 μg/day +GM-CSF 500 μg/day for 6 wks. ABLC, 5-FC, and fluconazole stoppedFavorable response Day 30 1 yr after EOT: allogeneic BMT for recurrent leukemia; died with liver veno-occlusive disease without infection.
Patient 3 15/femaleAML Induction chemotherapyChronic disseminated candidiasis and sino-pulmonary mould infection6 mo ABCD (11 g), AMB (1.6 g) Itraconazole (1 mo) Fluconazole + 5-FC (1 mo)IFN-γ 50 μg/m2 + G-CSF 5 μg/kg × 3/wk. G-CSF given for 4 mo and IFN-γ for 5 mo. 5-FC stopped. Fluconazole decreased from 400 to 200 mg/dayFavorable response Day 7 4 yrs after EOT: alive and well without infection.
Patient 4 28/maleAML Induction chemotherapyChronic disseminated candidiasis2 mo AMB (0.2 g)-/-ABLC (> 15 g) + fluconazole (2 mo)IFN-γ 50 μg/m2 + GM-CSF 250 μg/day × 3/wk for 3 mo. Fluconazole increased from 400 to 800 mg/dayFavorable response Day 7 3 mo after EOT: no infection. Death from recurrent leukemia after allogeneic BMT.

Patient 2 is a 22-year-old male with acute lymphocytic leukemia who developed disseminated Trichosporon beigelii infection during disease remission induction chemotherapy with fever, multiple skin lesions, acute respiratory distress syndrome (ARDS), and multiorgan failure. The organism was recovered from a skin biopsy sample, blood cultures, and from bronchoalveolar lavage specimens. Despite recovery from neutropenia and treatment with AMB then ABLC plus fluconazole and GM-CSF, fever persisted and new splenic lesions developed as observed on CT scan images. 5-FC was added for 14 days and he underwent a splenectomy. Histopathologic evaluation yielded fungal granulomas and T. beigelii infection. One month later, he developed infection-related hypercalcemia and fungal endophthalmitis, leading to the resumption of 5-FC and the intraocular administration of AMB for an additional month but without resolution of fever, pulmonary infiltrates, endophthalmitis, and hypercalcemia. The patient was considered refractory to 3 months of antifungal therapy including 21 g of ABLC, 34 days of 5-FC, and 3 months of fluconazole. All antifungal agents were discontinued and cytokine therapy was initiated with 100 μg/d IFN-γ plus 500 μg/d GM-CSF. Within the first 30 days of therapy, a clear response was observed with defervescence and resolution of fever, endophthalmitis, hypercalcemia, and pulmonary lesions. A second course of antineoplastic chemotherapy was given without recurrence of infection. One year later, the patient underwent matched related donor allogeneic bone marrow transplantation (MRD BMT) because of recurrent leukemia and he died 3 months after transplantation with veno-occlusive disease of the liver but without clinical or laboratory evidence of fungal infection. An autopsy was not performed.

Patient 3 is a 15-year-old female with AML who developed CDC by Candida tropicalis and sinopulmonary mold infection (organism present on histopathology but without growth) after neutropenia related to an MRD BMT. This patient with CDC presented with candidemia, and kidney and liver lesions as observed on CT scan images. Treatment with amphotericin B colloidal dispersion (ABCD) plus G-CSF–elicited granulocyte transfusions was started with resolution of kidney lesions, stabilization of hepatic lesions, but with persistence of sinopulmonary mold infection. Because of acute and chronic graft versus host disease (GVHD), she received maintenance therapy with AMB (total of 1.6 g) then ABCD (11.3 g). While receiving ABCD, she developed asthenia, a low-grade fever, infection-related hypercalcemia, and necrotic lymphadenitis by Candida spp. ABCD was discontinued and oral itraconazole was given for 1 month followed by another month of fluconazole plus 5-FC without response (fever, asthenia, hypercalcemia, purulent lymphadenitis, and recurrence of kidney lesions). She was considered refractory to 8 months of antifungal therapy. 5-FC was discontinued, the fluconazole daily dose was reduced from 400 mg to 200 mg, and cytokine therapy was initiated with 50 μg/m2 IFN-γ plus 5 μg/kg G-CSF 3 times a week. Within 7 days of cytokine therapy, the patient had a remarkable response with resolution of fever, hypercalcemia, anorexia, and asthenia and cessation of drainage at the site of lymphadenitis. Fluconazole was discontinued 2 months after the start of cytokine therapy and she continued to receive G-CSF and IFN-γ for a total of 4 and 5 months, respectively. A repeat CT scan after 4 months of cytokine therapy revealed stable kidney lesions but significantly larger liver lesions. Because of the absence of any clinical or laboratory evidence of infection, and the excellent performance status of the patient, no additional treatment was given. Four years after the EOT, the patient was free of infection despite persistence of chronic GVHD.

Patient 4 is a 28-year-old male with AML who developed CDC during chemotherapy-related neutropenia with fever, ARDS, renal failure, increase serum levels of AP and bilirubin, and liver and spleen lesions as observed on magnetic resonance imaging and CT scan images. Despite recovery from neutropenia and treatment with AMB followed by ABLC (total dose > 15 g) plus fluconazole for 2 months, he continued to have fever, anorexia, bilateral pulmonary infiltrates, and multiple liver and splenic lesions. A lung biopsy revealed granulomatous inflammation, and inflammation and fibrosis were observed on the fine-needle aspiration biopsy liver specimens. ABLC was discontinued, the fluconazole daily dose was increased from 400 mg to 800 mg, and cytokine therapy was initiated with 50 μg/m2 IFN-γ plus 250 μg/d GM-CSF 3 times a week. A clear response was observed 7 days after beginning cytokine therapy with a dramatic improvement in his performance status and anorexia but he developed purulent inguinal lymphadenitis on Day 14 of cytokine therapy. The lymphadenitis was drained surgically, and histopathologic evaluation revealed inflammation, necrosis, and yeast infection. On Day 21 of therapy, a CT scan revealed resolution of the pulmonary infiltrates but an increase in the number of hepatic and splenic lesions as his clinical condition continued to improve. Fluconazole and cytokines were discontinued 1 week and 3 months later, respectively, and the patient underwent a matched unrelated BMT. Despite the development of acute GVHD and the administration of high doses of corticosteroids, he remained free of fungal infection 3 months after discontinuation of cytokine therapy but died of recurrent leukemia. An autopsy was not performed.

These 4 patients responded promptly (as early as 7 days) to cytokine therapy and the response in Patients 3 and 4 was associated with an inflammatory reaction (increase in size and/or number of hepatic and splenic lesions, suppurative lymphadenitis). Patients 2, 3, and 4 were subsequently challenged with additional immunosuppression after discontinuation of cytokine therapy (allogeneic BMT [Patients 2 and 4] and chronic GVHD [Patient 3]) without recurrence of fungal infection. It is noteworthy that all antifungal agents were discontinued in Patients 1 and 2 at the start of cytokine therapy because of persistent infection despite several months of appropriate antifungal therapy. Cytokine therapy was well tolerated without dose reduction or drug discontinuation because of toxicity. Patient 3 who had active and chronic GVHD at the time of initiation of cytokine treatment did not have exacerbation of GVHD.

DISCUSSION

The current, small pilot study describes the response of four patients with leukemia with refractory invasive yeast infections to cytokine treatment, including two patients who responded to therapy with cytokines alone. Three patients received GM-CSF (Patients 1, 2, and 4) and 1 patient received G-CSF (Patient 3) in combination with IFN-γ. We did not use GM-CSF in Patient 3 because of the presence of chronic GVHD and concerns that the combination of two proinflammatory cytokines (IFN-γ and GM-CSF) might exacerbate GVHD.

During cytokine therapy, all patients were in complete disease remission, and had normal neutrophil counts for ≥ 1 month, no reduction in immunosuppressive therapy, and no addition of antifungal agents. Thus, response of their fungal infection was not associated with any of these parameters. In addition, the three patients who underwent subsequent immunosuppression remained free of fungal infection at last follow-up. Furthermore, the earlier response (within 7 days of initiation of cytokine therapy) observed in the 2 patients who exhibited findings compatible with a strong inflammatory reaction (Patients 3 and 4) may suggest a contribution of cytokines to the control of the infection.

Our findings support the favorable results observed in case reports of refractory IFI (Table 2). These reports described combination therapy with antifungal agents and either IFN-γ2–8 or GM-CSF,9 given to four patients with chronic granulomatous disease (CGD),2–4, 7 three patients with acute leukemia,5, 6, 9 and an otherwise healthy patient.8 Our results differ from previous reports in that antifungal treatment was discontinued in two patients, thus permitting the evaluation of the relative contribution of cytokine therapy alone to response and the use of a combination of IFN-γ plus a CSF.

Table 2. Patients with Refractory Fungal Infections Treated with Cytokines: a Literature Review
Patient Age (yrs)/gender DiseaseFungal infectionCytokine therapyOutcome after cytokine therapy
  1. 5-FC: 5-flucytosine; ABCD: amphotericin B colloidal dispersion; ABLC: amphotericin B lipid complex; ALL: acute lymphocytic leukemia; AMB: amphotericin B deoxycholate; AML: acute myeloid leukemia; BMT: bone marrow transplantation; CDC: chronic disseminated candidiasis; CGD: chronic granulomatous disease; CRP: C-reactive protein; CT: compute tomographic scan; D/C: discontinued; EOT: end of treatment; ESR: erythrocyte sedimentation rate; G-CSF: granulocyte—-colony- stimulating factor; GM-CSF: granulocyte-macrophage—-colony-stimulating factor; GVHD: graft vs. host disease; IFN-γ interferon-gamma; L-AMB: liposomal amphotericin B; mo: month; wk: week; qod: every other day; TD: total dose.

22/female, CGD2Pneumonia and bronchopleural fistula by Aspergillus-fumigatus untreated for 2 ys. Not candidate for surgery. Refractory to 6-mo AMB + short-course 5-FC. (+) Clinical findings + increased ESR) and (+) uptake in right lung, chest wall, ribs by 67Ga scan.IFN-γ (μg/m2 × 3/wk): 50 × 3 mo, 100 × 2 mo, 150 × 21 mo. AMB 10 mo, then, itraconazoleMo 8: marked decreased 67Ga scan accumulation and increased ESR. Mo 26: stable.
10/male CGD3Disseminated Pseudallescheria boydii (lungs, scalp, spine, spinal wound) refractory to 4 mo of miconazole, 3 mo of ketoconazole, and surgery on thoracic and lumbar vertebrae. Fever + spinal wound abscess + emesis + increased respiratory rate.IFN-γ 50 μg/m2 × 3/wk lifelong. Miconazole × 22 days. Itraconazole lifelong.Mo 4: clinical, microbiologic, and radiologic improvement. 4 yrs after EOT: no infection.
10/male CGD4Pneumonia and osteomyelitis by A. fumigatus. No response to 5 wks AMB. Fever, radiologic evidence of progression of osteomyelitis + increased ESR and increased CRPIFN-γ 50 μg/m2 × 3/wk × 6 wks. AMB stopped, itraconazole lifelong.Wk 5: afebrile, improvement of pneumonia and osteomyelitis, decreased. ESR and CRP. Mo 18 after EOT: asymptomatic
20/male ALL5Hepatosplenic Blastoschizomyces capitatus infection. No response to 3.5 mo AMB then 30 g ABLC. Abdominal pain + increased size of liver lesions on CT scan.IFN-γ 54 μg/m2 qod × 4 wks, then daily × 2 wks. Continued ABLC × 6 wks.Wk 1: clinical improvement. Wk 3: CT scan: increased liver lesions. Wk 6: resolution of liver lesions. Wk 7 after EOT: recurrent leukemia. No infection recurrence.
26/female, CGD7Multiple recurrent brain abscesses by A. fumigatus despite 8 surgical procedures, AMB, and itraconazole.IFN-γ 50 μg/m2 × 3/wk, itraconazole.No additional disease recurrences.
14/male, AML post chemotherapy6Pneumonia by Aspergillus spp. progressed despite recovery from neutropenia, AMB and L-AMB. Recurrent cerebellar abscesses. Poor performance status, increasing CRP, hypoxia.IFN-γ 60 μg/m2/day × 10 days. L-AMB continued.Day 4: decreased CRP. Day 14: resolution fever, hypoxia. Day 18: chemotherapy without infection recurrence. AML recurrence and death. No infection.
57/female, AML post chemotherapy9Disseminated Blastoschizomyces capitatus infection (blood, lungs, kidneys, spleen, liver, and central nervous system). Persistence of clinic radiologic signs of infection after 3 mo AMB (TD 2.2g) + 5-FC.GM-CSF 300 μg 3/wk × 4 wks, then, 150 μg × 2/wk × 16 wks. AMB + 5 FC × 4 wks, then, 5-FC × 16 wks.Wk 4: Marked decreased abdominal, pulmonary, and neurologic abscesses. Mo 11 after EOT: alive. No infection.
57/female, Healthy8Disseminated coccidioidomycosis after receipt of mechanical ventilator care for respiratory failure despite 10 wks of antifungal treatment that included ABLC, L-AMB + fluconazole, and L-AMB + itraconazole.IFN-γ 1b 50 μg/m2 × 3/wk for 9 wks. L-AMB continuedMo 1: improvement. Extubated and transferred to a rehabilitation center with receipt of fluconazole.

Acute leukemias and their treatments can impair the function and number of phagocytic and nonphagocytic cells directly or through the modulation of cytokine production.1, 10 Neutrophils, monocytes, T cells, and macrophages are critical for the suppression and killing of Aspergillus spp. and Candida spp. IFN-γ enhances the function of both neutrophils and monocytes against fungi, increases the hyphal damage mediated by phagocytes against several fungi, and shifts the cellular-mediated immune response from a Th2 to a Th1 pattern.1 This cytokine has also been shown to enhance the ability of neutrophils to damage Aspergillus hyphae ex vivo in patients with CGD,11 and to accelerate the sterilization of the cerebrospinal fluid in human immunodeficiency virus (HIV)-infected patients with cryptococcal meningitis.12 Two of our patients (Patients 3 and 4) had worsening of the imaging studies (liver, kidneys, or spleen) and 1 of these 2 developed lymphadenitis while clinically improving. IFN-γ has been shown to be crucial for the development of granulomas13 in animal models. It is possible that these patients were unable to mount an adequate inflammatory response because of severe immunosuppression, and that immune reconstitution with cytokines allowed the formation of granulomas that could then be more clearly delineated on imaging studies. This phenomenon might be similar to that observed in HIV-infected patients receiving highly active antiretroviral therapy (HAART). In these patients, subclinical infections may be unmasked by the enhancement of their immune system with HAART.14

The CSFs have also been shown to enhance the in vitro antifungal activities of phagocytes against opportunistic fungi.1 An in vitro additive antifungal effect was also observed with the combination of IFN-γ and G-CSF and the combination of any of these cytokines with antifungal agents.15

Our study is limited by the small cohort of patients, its retrospective nature, and the lack of supporting laboratory studies. It could also be argued that the favorable outcome was due to the concomitant antifungal agent. This is unlikely, given the discontinuation of antifungal therapy in two patients and the continuation of the same agents in the other two patients who had progressive infection. In addition, persistent infection before commencing cytokine therapy was well documented in all patients, despite prolonged antifungal therapy and recovery from neutropenia.

We conclude that our pilot study suggests a potential role for immunomodulation in patients with cancer with refractory IFI. Based on our study, we cannot recommend any specific timing for the initiation of cytokine therapy but rather suggest this modality when there is clear evidence of failure to respond to appropriate antifungal agents in a patient with adequate neutrophil counts and remission of underlying leukemia.

Ancillary