G-CSF withdrawal-induced apoptosis of neutrophils following systemic chemotherapy for patients with recurrent breast cancer

Y Ogawa, MD, PhD a, T Kobayashi, PhD b, A Nishioka MD, PhD a, S Kariya MD, PhD a, H Seguchi MD, PhD b, S Yoshida MD, PhD a

Departments of aRadiology and bAnatomy & Cell Biology , Kochi Medical School, Nankoku, Kochi, Japan

The effectiveness of granulocyte colony-stimulating factor (G-CSF) for neutropenia has been widely recognized in the field of systemic chemotherapy. However, although neutrophils counts rapidly increase after a few times injections of G-CSF, a large proportion of the increased neutrophils disappear from peripheral blood in a few days following G-CSF withdrawal. We intended to clarify the origin of the early decrease of increased neutrophils following G-CSF withdrawal for patients with recurrent breast cancer. After obtaining informed consent, neutrophils were isolated from the peripheral blood of ten patients with recurrent breast cancer undergoing systemic chemotherapy. Changes in the mitochondrial membrane potential of neutrophils were examined using a CCD camera-equipped fluorescence microscope and MitoCapture Mitochondrial Apoptosis Detection Kit (BioVision, USA). MitoCapture that has aggregated in the mitochondria of non-apoptotic neutrophils fluoresces red. In apoptotic neutrophils, MitoCapture cannot accumulate in the mitochondria but only the contours of neutrophils can be recognized. Here we demonstrate a representative case, because similar results were obtained for another nine patients. A patient with recurrent breast cancer was injected with 5 microgram/kg of G-CSF subcutaneously for five consecutive days, in order to treat her neutropenia which had fallen to approximately under 500 following previously performed docetaxel-based chemotherapy. Approximately 30-40% of the neutrophils were identified to be apoptotic throughout the next day to the second day after the sudden termination of G-CSF injection. On the third day, the percentage of apoptotic neutrophils decreased to 15%. On the fourth day, her neutrophil counts became stable at 3000, and almost all neutrophils were identified to be non-apoptotic. At the second course of the systemic chemotherapy with the same regimen, the dose of G-CSF injection was tapered down to 2 microgram/kg and to 1 microgram/kg after the occurrence of WBC counts of 5000/microliter after the neutrophil nadir. Apoptotic neutrophils were hardly seen throughout the period. Using MitoCapture and a CCD camera-equipped fluorescence microscope, we succeeded in demonstrating that G-CSF withdrawal induced loss of mitochondrial membrane potential, i.e., an early stage of apoptosis, in human peripheral neutrophils increased by G-CSF for patients with recurrent breast cancer. Therefore, there is an urgent need to change G-CSF use from the sudden withdrawal method currently used to a new method of tapering down the dose in order to avoid excess apoptotic loss of the increased neutrophils. Such a simple observation helps both to establish the appropriate use of G-CSF for each individual patient and to reduce the medical costs of cancer chemotherapy.

KEY WORDS: breast cancer, chemotherapy, neutrophil, granulocyte, colony stimulating factor.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.

This presentation received an honorable mention in our poster contest and was recognized with the Symposium Presidents' Award for Scientific Excellence.