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© 2002 American Society for Clinical Oncology Reinfusion of Autologous Lymphocytes With Granulocyte-Macrophage Colony-Stimulating Factor Induces Rapid Recovery of CD4+ and CD8+ T Cells After High-Dose Chemotherapy for Metastatic Breast CancerByFrom the Department of Medical Oncology, Department of Immunology, and Department of Clinical Chemistry, The Netherlands Cancer Institute, Amsterdam, the Netherlands. Address reprint requests to G.C. de Gast, MD, PhD, Department of Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands; email: p.favre{at}nki.nl
PURPOSE: Repeated high-dose chemotherapy (HDCT) followed by peripheral-blood progenitor cell (PBPC) transplantation can induce a complete remission in patients with metastatic breast cancer sensitive to standard chemotherapy (CT), but the majority of patients relapse within 1 to 2 years. The immune system is seriously compromised after HDCT, which precludes the development of effective immunotherapy. We investigated whether autologous lymphocytes, reinfused after HDCT, could induce a rapid recovery of T cells. PATIENTS AND METHODS: Three patients were monitored for immune recovery without reinfusion of lymphocytes. In the next 11 patients, stem cells were harvested after CT + granulocyte colony-stimulating factor (G-CSF) and lymphocytes were harvested after CT + granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2. These patients received stem cells and G-CSF after the first HDCT; stem cells, G-CSF, and lymphocytes after the second; and stem cells, GM-CSF, and lymphocytes after the third HDCT. RESULTS: Patients not receiving lymphocyte reinfusion had a very slow recovery of lymphocytes. In particular, CD4 counts remained low (< 200/µL for 9 months). Lymphocyte reinfusion had a significant effect on the recovery of lymphocytes, T cells, and CD8+ T cells (normalized on day 25). Recovery of CD4+ T cells was significantly accelerated by lymphocyte reinfusion and GM-CSF, leading to counts of 500/µL at 25 days. CONCLUSION: Lymphocyte reinfusion with G-CSF had a significant effect on the recovery of CD8+ T cells, whereas rapid recovery of CD4+ T cells required lymphocyte reinfusion and GM-CSF, which possibly acts as a survival factor through activation of antigen presenting cells. Whether the rapid recovery of CD4+ and CD8+ T cells prevents or delays relapse of the disease should be further investigated.
METASTATIC BREAST cancer generally is incurable with standard chemotherapy (CT). With repeated cycles of high-dose chemotherapy (HDCT) followed by reinfusion of autologous stem cells, complete remissions can be induced in the majority of patients responding to anthracycline-based CT.1-5 However, only a small minority remains free of disease. HDCT also induces profound immune deficiency, which is associated with recurrent bacterial and viral infections such as varicella zoster virus (VZV). It is conceivable that severely impaired antitumor immunity also contributes to relapse of the disease after achieving minimal residual disease status. Much has been learned about immune recovery after human stem-cell transplantation from allogeneic bone marrow transplantation with or without T-cell depletion.6-7 Recovery of natural killer (NK) cell number and function is generally complete within 1 month and a result of differentiation from stem cells. B cells recover more slowly, mainly by differentiation from stem cells and direct precursors. The situation for T cells is completely different; CD4+ T cells of the memory type recover only by peripheral expansion in adults, which takes several months. In children, recovery of the naive type (CD4+ CD45RO) is dependent on the presence of a thymus, which is related to age.8-10 The subset of CD8+ T cells generally recovers much faster because of combined peripheral expansion and extra-thymic differentiation from stem cells.6-10 Especially functional recovery of T and B cells is influenced by graft-versus-host disease, a component not present after autologous marrow or peripheral-blood stem-cell transplantation.6 Functional CD4+ T-cell recovery has been shown to be mainly a result of peripheral expansion.6-11 Little is known how these T-cell deficiencies can be corrected, but data on donor leukocyte infusions after allogeneic bone marrow transplantation show that this can be associated with rapid restoration of CD3+, CD4+, and CD8+ T-cell numbers, inclusive antigen-specific T-cell responses.12 If this is true, recovery of CD4+ T (and CD8+ T) cells could be achieved by the reinfusion of autologous lymphocytes, harvested before HDCT. We surmised that a rapid recovery of the immune system in a situation of minimal residual disease created by HDCT is a prerequisite for the design of effective immunotherapy. This is contrary to current trends of purifying CD34+ stem cells because of the risk to reinfuse circulating tumor cells, which are often present in bone marrow and peripheral blood even in solid tumors and have been suggested to correlate with relapse.13,14 In this article, we show that activated autologous lymphocytes harvested after standard CT followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose interleukin (IL)-2 can rapidly restore T-cell deficiencies.
Patients with histologically proven epithelial breast cancer with distant metastases (M1) were eligible, if they had hormone refractory disease (defined as estrogen receptor–negative or had failed at least one adequate hormonal therapy in case of estrogen receptor–positive tumor) and never had anthracycline-containing CT. Patients were aged  55 years, had a performance status 0 or 1 (Zubrod scale), and had normal renal, liver, and bone marrow function, were negative for human immunodeficiency virus antibodies and hepatitis B antigens, and signed informed consent. Patients with autoimmune disease previously or presently were excluded as well as patients with severe cardiac, pulmonary, or metabolic disease. The CT regimens have been described previously.4-5 Patients were treated with two cycles of fluorouracil 500 mg/m2, epirubicin 120 mg/m2, and cyclophosphamide 500 mg/m2 (FEC) intravenously every 3 weeks. After the second course of FEC and granulocyte colony-stimulating factor (G-CSF; filgrastim) (300 µg subcutaneously irrespective of body weight), peripheral-blood progenitor cells (PBPC) were harvested to support three cycles of HDCT. At least 3 x 106/kg CD34+ cells were considered adequate per cycle HDCT. If the patients had evidence of a response, a third FEC course was given followed by GM-CSF (molgramostim) 2.5 µg/kg for 12 days and, in the last 7 days, low-dose interleukin-2 (IL-2; aldesleukin) 2 MIU/m2 subcutaneously. The second day after stopping GM-CSF and IL-2, peripheral-blood mononuclear cells (PBMC) were harvested (aimed at a total number of 2 x 1010) and frozen in liquid nitrogen (in 10% DMSO, freezing technique similar to that used for stem cells), to be thawed and infused after the second and third HDCT.4 The reason for giving a third FEC course followed by GM-CSF and low-dose IL-2 was primarily to harvest activated autologous lymphocytes. However, we assumed that by giving GM-CSF after CT, tumor cell antigens released from dying tumor cells would be presented by activated dendritic cells to T cells, possibly leading to T-cell reactivity to the tumor in the harvested T cells by the combined action of GM-CSF and low-dose IL-2. Thus, the difference between the stem-cell preparation and the activated lymphocytes used for reinfusion was that the lymphocytes/PBMC were harvested on the second day after stopping GM-CSF and IL-2. HDCT consisted of carboplatin 1,100 mg/m2, thiotepa 320 mg/m2, and cyclophosphamide 4,000 mg/m2 (tiny [t]CTC) intravenously, divided over 4 days.4,5 As shown in Fig 1, all three tCTC cycles were supported by reinfusion of autologous stem cells. After the first cycle, G-CSF was given until neutrophil recovery; after the second cycle, G-CSF and autologous PBMC were given; and after the third cycle, GM-CSF and autologous PBMC were given. By this procedure, we could compare the influence of reinfusing T cells combined with G-CSF (second tCTC) and with GM-CSF (third tCTC) to no infusion of T cells (first tCTC) in the same patient. After platelet recovery more than 50 x 109/L, IL-2 (2 MIU/m2 subcutaneously) was given for 12 days in the second month after the third tiny CTC.
Flow Cytometry To detect cellular differentiation markers on peripheral-blood cell samples taken before, during, and after immunotherapy, WBCs were collected from heparinized peripheral blood by lysis of red cells with ammoniumchloride. Triple staining was performed by incubation of cell samples in a mixture of fluorescein (FITC)-, R-phycoerytrin (PE)-, and PE-cychrome (Cy5)-conjugated mouse monoclonal antibodies, directed to cellular differentiation markers, for 20 minutes at room temperature followed by washing in PBS containing 0.2% BSA and 0.02% azide, as described previously.15 Monoclonal antibodies used were as follows: FITC- and PE-conjugates from Becton & Dickinson Immunocytometry Systems, San Jose, CA (aCD3-FITC + CD [16 +56] PE; aCD4-FITC; aCD8-FITC; aCD14-FITC; and aHLA-DR-PE) and Cy5-conjugates from Coulter Immunotech, Mijdrecht, the Netherlands (aCD19-PC5; aCD3-PC5; and aCD15-PC5). To prevent aspecific binding via Fc receptors, incubation was carried out in the presence of 1% normal mouse serum (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands). Fluorochrome-labeled isotype control antibodies were included in each assay to determine background staining. Lymphocyte (CD3+, CD19+, and CD16+56+) and monocyte (CD14+) populations were gated according to their differential forward and side scatter. Fluorescence was measured on a FACScan flow cytometer (Becton & Dickinson, Mountain View, CA), and data were analysed using Cellquest software (Becton & Dickinson).
Statistics
The first three patients received three tCTC without lymphocyte reinfusion after two cycles of FEC. As shown in Fig 2A, they had a very slow recovery of lymphocytes, reaching normal levels after 1 year. T cells (CD3) never reached normal levels with a maximum of 600/µL after 9 months and were generally activated (HLA-DR positive). NK cell recovery was adequate after 2 months and B-cell recovery was slow. As listed in Table 1, the impaired T-cell recovery was mainly a result of impaired CD4+ T-cell recovery, reaching the low level of 200/µL only after 9 months. CD8+ T-cell recovery was slightly better, with levels of 200/µL after 2 months.
The next 11 patients had a third FEC, followed by GM-CSF + low-dose IL-2, and we aimed to harvest 2 x 1010 lymphocytes. Actually, 1.6 to 4.8 x 1010 lymphocytes were harvested through one to two phereses and reinfused (Table 2). Toxicity of concurrent GM-CSF and low-dose IL-2 consisted of chills, fever, and flu-like symptoms, as previously described.15 One patient with allergic asthma needed inhalation corticosteroids for control of symptoms. All patients had one or two metastatic sites and had a response to FEC (except patient no. 7). Four patients received only two tCTC (patient nos. 1 to 4), with lymphocyte-reinfusion + G-CSF after the second tCTC, because of patient refusal to continue therapy, severe allergic reactions to thiotepa, anaphylactic reaction to the second tCTC, or development of veno-occlusive disease. Seven patients (patient nos. 5 to 11) received three courses of tCTC with lymphocyte reinfusion + GM-CSF after the third tCTC. All patients except one (10 of 11 patients) achieved complete response after two or three tCTC without additional therapies. All 11 patients had limited metastatic disease, consisting of soft tissue metastases including lymph nodes and one patient with liver and bone metastases (no. 7) and one with lung metastases (no. 8) (Table 2). The patient with liver and bone metastases had stable disease on both metastatic sites after FEC and three tCTC. Follow-up is 12 to 27 months after the start of the first FEC (median, 21 months). Five patients relapsed 6 to 16 months after starting, and one patient with stable disease after HDCT had progression after 14 months. One of these patients died after 8 months from cerebral metastases; five of seven patients receiving three tCTC remained tumor-free. The four patients receiving two tCTC and reinfusion of lymphocytes after the second tCTC, followed by G-CSF, had an impaired slow CD4 T-cell recovery not significantly different from the first three patients (prestudy group) (Table 1). However, CD8+ T-cell recovery was significantly faster and higher than in the first group.
As shown in Fig 2B, the seven patients receiving three tCTC, lymphocyte reinfusion, and GM-CSF had a good lymphocyte, T-cell, and NK cell recovery up to pretreatment levels at 25 days after the second and third tCTC, which was significantly better than after the first tCTC (P < .05) for lymphocytes and the prestudy group (P < .01). B-cell (CD19+) levels recovered slowly and were comparable with the prestudy patients. In Fig 3, CD4 and CD8 T-cell recovery is depicted in the patients receiving three tCTC. For CD4+ T cells, levels of more than 500/µL were reached after the third tCTC, which was significantly higher (P < .05) than after the first and second tCTC. CD4+ T-cell levels remained 300 to 400/µL during follow-up. CD8+ T-cell recovery showed a different picture with a rapid recovery after the second and third tCTC, up to levels higher than pretreatment levels and higher than after the first tCTC but without a difference between the second and third CTC. CD8+ T-cell levels remained high (approximately 500/µL) for 9 months.
Analysis of the relationship between the number of infused cells and recovery after the second (patient nos. 1 to 4) or third tCTC (patient nos. 5 to 11) (Table 3) showed that such a relationship was present for recovery of lymphocytes and T cells. Patients receiving two tCTC + G-CSF reached significantly lower numbers of CD4+ T cells at 1 month than patients receiving three tCTC + GM-CSF (median, 84/µL v median, 495/µL; P < .05). In contrast, CD8+ T-cell levels were not significantly different (448/µL v 589/µL). Recovery of CD8+ T cells showed a significant relationship with the number of infused CD34+ cells (P .005); low numbers of reinfused CD34+ cells (< 5.0 x 106/kg) resulted in poor NK cell recovery in two patients (nos. 1 and 6). Six of 11 patients showed a VZV infection, whereas all patients were prone to reactivation (immunoglobulin G antibody positive) (Table 3). Patients not showing a reactivation were characterized by rapid NK cell recovery (> 300/µL in 1 month; patient nos. 7, 8, and 11) or CD8+ T-cell recovery (> 500/µL in 1 month; patient nos. 2, 3, and 7).
In this study, we addressed the question whether immune deficiency caused by HDCT could be prevented by reinfusion of autologous lymphocytes. First, we documented how profound the immune deficiency is after two cycles of standard CT (FEC) followed by three cycles of HDCT (tCTC) supported by stem cells and G-CSF in patients with metastatic breast cancer. Previously, others also described a serious dysfunction in cellular immunity after stem-cell transplantation for advanced breast cancer that persisted in a majority of patients for at least 6 months or until disease progression.16 And, indeed, the immune deficiency was profound and of long duration with lymphocytes reaching normal values only after 1 year, whereas T cells, especially CD4+ T helper cells remained very low at levels comparable with those seen in AIDS (< 200/µL). In contrast, NK cells recovered after 2 months, and B cells recovered after 9 months. Lymphocytes harvested after a third cycle of standard CT (FEC) that was followed by GM-CSF and low-dose IL-2 were indeed capable to induce a rapid restoration of lymphocytes and T cells (both CD4+ and CD8+ T cells) if reinfused after the third tCTC and followed by GM-CSF. Remarkably, no difference was seen in the recovery of B cells, and only a marginal difference was seen in that of NK cells (full recovery after 1 month instead of after 2 months). A subgroup of four patients received only two tCTC courses followed by reinfusion of stem cells, lymphocytes, and G-CSF and showed good recovery of lymphocytes and CD8+ T cells, which is probably because of the reinfusion of lymphocytes, although an influence of a lower cumulative dose of CT (two courses of tCTC) cannot be excluded. In contrast, CD4+ T cells recovered only slightly better than in the first three patients who had not received reinfusion of lymphocytes. This also suggests that GM-CSF is an important factor for survival/recovery of CD4+ T cells. Because seven patients received three courses of tCTC, an intrapatient comparison could be performed in this group on the effect of lymphocyte reinfusion and the effect of G-CSF versus GM-CSF. This study showed the same results as comparison with the prestudy group without reinfusion and with the patients receiving only two tCTC with G-CSF. Lymphocyte reinfusion had a profound effect (comparison first and second tCTC) on recovery of CD8+ T cells, but for recovery of CD4+ T cells, GM-CSF was required as well (comparison second and third tCTC). A low recovery of NK cells was found in the two patients receiving less than 5.0 x 106/L CD34+ stem cells, and a significant correlation was found between the number of reinfused CD34+ cells and recovery of CD8+ T cells despite the fact that all patients received lymphocyte reinfusion. These data confirm the notion that B cells and NK cells are mainly or completely derived from stem cells and do not need the thymus for development. CD8+ T cells are partly dependent on reinfused T cells and partly on the number of CD34+ cells, probably via differentiation through an extra-thymic pathway. CD4+ T cells, in contrast, need a functional thymus as is clearly shown in children treated with HDCT.8 In adults without a functional thymus, as in our patient group, T-cell recovery is mainly dependent on mature T cells that are reinfused together with stem cells or separately as in our study. The same has been noted after autologous stem-cell transplantation for hematologic tumors.11 Recently, it has been described that in patients with multiple myeloma thymic function can recover after myeloablative CT as detected by T-cell receptor rearrangement excision circles and CD45 RA+ CD4+ T cells.17 Whether thymic recovery also does occur in breast cancer patients who have had irradiation of the breast is not clear. A new finding in our study was that reinfused CD4+ T cells obtained after CT + GM-CSF and low-dose IL-2 do need GM-CSF in addition to reinfusion for a good and rapid recovery, because reinfusion of CD4 + T cells followed by G-CSF after the second CTC did not lead to a rapid recovery. Previously, it has been shown that GM-CSF can cause T-cell activation.18-20 The effect of GM-CSF probably is indirect via production and activation of dendritic cells, which are known to have a profound effect on survival and proliferation of CD4+ T cells, mainly via CD40, B7-1, and B7-2 (CD80/CD86) expression on dendritic cells and CD40 L and CD28 on CD4+ T cells.21-22 Preliminary results showed that the mononuclear cells harvested after standard CT followed by GM-CSF and IL-2 contained more CD14+/CD40+ cells with also a higher expression of CD40 compared with initial samples (results not shown). In this respect, it will be interesting to know whether GM-CSF after each cycle of HDCT can rescue CD4+ T cells without reinfusion of lymphocytes. Of course, the most important question is whether a rapid recovery of CD4+ and CD8+ T cells has clinical consequences not only for prevention of infection but especially for prevention of relapse. In our study, VZV infection still occurred in six of 11 patients. Such prevention may be dependent on rapid recovery of NK cells as observed in three patients (nos. 7, 8, and 11) or on the presence of VZV antigen-specific T cells, which was not studied, but suggested by high CD8+ T-cell recovery (patient nos. 2, 3, and 7). Considering relapse of the disease, it is noteworthy that five of seven patients receiving three courses of tCTC, and stem-cell and lymphocyte reinfusion, followed by GM-CSF are still relapse-free after 12 to 27 months of follow-up (median, 21 months). The patient with progression after 15 months had stable disease after HDCT. One patient relapsed after 16 months, whereas the five other patients remained in complete response. These patients all had a rapid recovery of CD4+ and of CD8+ T cells within 1 months after the last tCTC. From these data, it seems likely that reinfusion of autologous lymphocytes does not increase the risk of relapse but possibly even helps to prevent it. If true, this would cast serious doubt on the desirability of purging CD34+ stem cells to get rid of tumor cells, as theorized by some.23 Whether the low-dose IL-2 that was given in the second month after full recovery of the platelets had any influence remains unclear. If CD4+ T cells are essential for the prevention of relapse, it has to be established whether this is a direct effect of CD4+ T cells (by cytokine release or cytotoxicity) or indirect via activation and differentiation of CD8+ T cells, B cells, or NK cells. Thus, rapid recovery of CD4+ and CD8+ T cells after high-dose CT is possible by reinfusion of autologous lymphocytes and administration of GM-CSF. HDCT with rapid recovery of the immune system may be a new promising modality that should be studied further.24,25
We thank Irene Urlus for secretarial assistance.
Immunomonitoring and interleukin-2 was supported by Chiron Therapeutics, Amsterdam, the Netherlands.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
in progressive metastatic melanoma and renal cell carcinoma. Clin Cancer Res 6: 1267-1672, 2000
