This study evaluated the effects of low-dose IL-2 plus G-CSF/EPO on post-PBSC transplantation (PBSCT) immune-hematopoietic reconstitution and NK activity in patients with breast (BrCa) and ovarian cancer (OvCa). To this end, two consecutive series of patients were prospectively assigned to distinct post-PBSCT cytokine regimens (from day +1 to day +12) which consisted of G-CSF (5 μg/kg/day) plus EPO (150 IU/kg/every other day) in 17 patients (13 BrCa and 4 OvCa) or G-CSF/EPO plus IL-2 (2 × 105 IU/m2/day) in 15 patients (10 BrCa and 5 OvCa). Hematopoietic recovery and post-transplantation clinical courses were comparable in G-CSF/EPO- and in G-CSF/EPO plus IL-2-treated patients, without significant side-effects attributable to IL-2 administration. In the early and late post-transplant period a significantly higher PMN count was observed in G-CSF/EPO plus IL-2-treated patients (P = 0.034 and P = 0.040 on day +20 and +100, respectively). No significant differences were found between the two groups of patients in the kinetics of most lymphocyte subsets except naive CD45RA+ T cells which had a delayed recovery in G-CSF/EPO plus IL-2 patients (P = 0.021 on day +100). No significant difference was observed between NK activity in the two different groups, albeit a significantly higher NK count was observed in G-CSF/EPO plus IL-2 series on day +20 (P = 0.020). These results demonstrate that low-dose IL-2 can be safely administered in combination with G-CSF/EPO early after PBSCT and that it exerts favorable effects on post-PBSCT myeloid reconstitution, but not on immune recovery.
Recent clinical trials have shown a possible role for induction chemotherapy followed by high-dose consolidation treatment in advanced ovarian cancer (OvCa) patients1,2 and high-risk breast cancer (BrCa) patients.3,4 Autologous PBSC transplantation (PBSCT) represents a supportive measure to rapidly reconstitute hematopoiesis following the administration of myeloablative high-dose chemotherapy. Hematopoietic growth factors such as G-CSF or GM-CSF accelerate neutrophil recovery with a consequent reduction in the number of days with fever and antibiotic treatment, without relevant effects on platelet and erythroid recovery. Theoretically, the clinical use of exogenous cytokines after PBSCT may cause a selective expansion of hematopoiesis in which a single lineage might expand at the expense of the others, via a mechanism of progenitor cell competition. This point assumes particular interest if we consider that post-chemotherapy immune-hematopoietic reconstitution may contribute to the control of the residual disease. With reference to this, recent data suggest that reconstitution of immune response could eradicate tumor cells that have escaped the cytotoxic damage produced by chemotherapy.5,6,7 Our previous data showed that both G-CSF and GM-CSF were equally effective in promoting myeloid engraftment following PBSCT, but G-CSF promoted a more valid immune recovery.8 Conceivably, the early administration of a growth factor involved in T cell development, such as IL-2, might produce a lymphoid-oriented differentiating stimulus on transplanted stem cells and activation on de novo generated lymphoid cells. To verify the role of post-PBSCT low-dose IL-2 administration, we carried out a prospective non-randomized study where G-CSF/EPO-treated patients were compared with G-CSF/EPO plus IL-2-treated patients at several time points from PBSCT. We report the results of our comparison with respect to hematopoietic recovery, clinical management, and immune recovery including NK function.
Patients and methods
Patient's eligibility and treatment plan
Twenty-three patients with resectable high-risk BrCa (stage II–IIIa with ⩾4 involved nodes) and nine patients with stage IIIb-c OvCa with a residual tumor <1 cm after primary cytoreductive surgery or interval debulking surgery (IDS),9 ranging in age from 33 to 63 years, were enrolled into this prospective phase II study investigating G-CSF/EPO and G-CSF/EPO plus IL-2 following high-dose chemotherapy with carboplatin (1200 mg/m2), etoposide (900 mg/m2) and melphalan (100 mg/m2) (CEM) and PBSCT.10 Prior to CEM administration, all patients were treated with induction nonmyeloablative chemotherapy which was also used to mobilize and collect PBSC, as previously described.11,12 None of the patients received radiotherapy. Eligibility criteria included a performance status of 0–2 (WHO scale), adequate pulmonary, cardiac, hepatic and renal function, absence of underlying infections, a WBC count >2000 per μl and a platelet count >100 000 per μl. The study was approved by the Hospital Human Investigation Review Board and written informed consent was obtained from all patients. Patient characteristics at the time of completion of nonmyeloablative chemotherapy are detailed in Table 1. Following CEM administration (day −4, −3, −2, and −1) and PBSCT (day 0) the first 17 consecutive patients received rhG-CSF (Neupogen; Dompe’ Biotec, Milan, Italy) 5 μg/kg/day subcutaneously from day +1 to day +12. The subsequent 15 patients received rhG-CSF 5 μg/kg/day subcutaneously from day +1 to day +12, plus concomitant IL-2 (Proleukin; Chiron, Emeryville, CA, USA) 200 000 IU/m2 subcutaneously from day +1 to +12. All patients received rhEPO (Eprex; Cilag, Milan, Italy) 150 IU/kg/every other day subcutaneously from day +1 to day +13 in order to maintain an adequate serum EPO level and to potentiate the effect of G-CSF, as described previously.11,13,14,15 Oral antibacterial, antifungal and antiviral prophylaxis was given according to current investigational protocols. All patients received a minimum dose of circulating CD34+ cells of 3.0 × 106/kg body weight. All were transfused with irradiated RBC when the hematocrit value was lower than 23% and with irradiated single-donor platelet concentrates when the platelet count was lower than 10 000/μl or during bleeding episodes. Empiric antibacterial therapy was started in the presence of fever >38.5°C lasting for more than 12 h and was continued until the disappearance of fever for more than 3 days and/or complete remission from a clinically or microbiologically documented infectious episode. Hematopoietic recovery was defined as the number of days necessary to reach 1000 WBC per μl of whole blood, 500 PMN per μl, 50 000 platelets per μl and 20 000 reticulocytes per μl. Following PBPCT, all patients were discharged from the hospital when the PMN count was >500/μl for 3 consecutive days and platelet count >50 000/μl, in the absence of fever, documented infections or relevant nonhematological toxicities.
Hematological and immunological monitoring
Complete blood counts and WBC differentials were taken daily in all patients during their hospital stay, required for CEM administration, PBSC infusion and post-transplantation clinical management of hematological and non-hematological toxicities. In both patient groups lymphocyte immunophenotyping with complete blood counts, WBC differentials and NK activity were determined on day +20, +60 and +100 to document the kinetics of early/late myeloid/immune recovery. Blood counts were performed from samples collected in EDTA using the Bayer Technicon H3 RTX System (Bayer Technicon, Tarrytown, NY, USA) and by cytological examination of blood smears for the assessing of WBC differentials during and after cytokine treatment. The variation coefficient (CV) of blood counts estimated by the Bayer Technicon H3 RTX System was 3.8% for WBC, 1.4% for hemoglobin and 6% for platelets. Reticulocyte counts were performed to document erythroid recovery by the dedicated and automated reticulocyte counter Sysmex R-1000 (Sysmex Toa, Kobe, Japan), as previously described.16
Lymphocyte immunophenotyping by flow cytometry
Immunophenotyping was performed on whole blood samples according to the procedures recommended in published guidelines.17 Monoclonal antibodies (mAbs; all from Becton Dickinson, San Jose, CA, USA) with the following specificity were used: CD3-, CD4-, CD45-, and CD45RA-fluorescein isothiocyanate (FITC); CD8-, CD14-, CD19-, CD56/16 and CD45RO-phycoerythrin (PE); CD3-peridinin chlorophyll protein (PerCP). Dual and three-color fluorescence was performed using the following combinations of mAbs: CD14/CD45; CD3/CD56/CD16; CD3/CD45RA/CD45RO; CD3/CD4/CD8; CD3/CD19. Concentration of antibodies was determined by titration and background staining assessed by isotype-matched fluorochrome-conjugated irrelevant mAbs. After staining, flow cytometric evaluation was performed immediately on unfixed cells on a FACSCan. Absolute counts of the various lymphocyte subsets were calculated by multiplying the percentage of cells positively identified by the various mAb combinations on absolute lymphocyte count.
Natural killer (NK) cell activity was assessed using a previously described technique that employs K562 cells as a target in a 4-h 51Cr release assay.18 Several effector/target ratios ranging from 6:1 to 50:1 in replicate wells of a 96-well microtiter plate were established from each mononuclear cell sample obtained from patient peripheral blood collected at the different time points by gradient isolation using Ficoll–Hypaque (Pharmacia LKB, Uppsala, Sweden; density 1.077 g/ml) and centrifugation at 400 g for 30 min at 20°C.18 Incubation lasted 4 h at 37°C. The specific lysis was determined according to the previously published formula:18
Spontaneous counts per min (c.p.m.) were determined by measuring the amount 51Cr released from 104 target cells incubated in the absence of effector cells. Maximal c.p.m. were determined by resuspending 104 target cells into the assay supernatant.
Comparisons between patient series were performed by Mann–Whitney U nonparametric tests. A P value <0.05 was considered significant.
Myeloid recovery and clinical management following PBSCT
Seventeen and 15 patients were enrolled in the G-CSF/EPO and G-CSF/EPO plus IL-2 group, respectively, and their characteristics at the time of high-dose chemotherapy are detailed in Table 1. T and B lymphocyte counts prior to PBSCT and the number of CD34+, CD3+ and CD19+ cells infused per kg of recipient weight were similar in the two groups (data not shown). Following high-dose chemotherapy and PBSCT, myelosuppression was complete in all cases with a WBC count below 50 per μl and platelet count below 10 000 per μl in most cases. All patients were evaluable for hematopoietic recovery, clinical care and follow-up. The kinetics of hematopoietic recovery, defined as days to reach 1000 WBC per μl, 500 PMN per μl, 50 000 platelets per μl and 20 000 reticulocytes per μl are shown in Table 2. Our results indicate that the number of days required to achieve blood cell recovery was comparable in both series and similar to that previously described in patients following CEM and PBSCT.10 In particular, more than 500 PMN/μl were reached after a median of 9 days in both groups, while a platelet count greater than 50 000/μl was reached after a median of 12 and 11 days in G-CSF/EPO- and G-CSF/EPO plus IL-2-treated patients, respectively. Similarly, erythropoietic recovery, documented by the presence of more than 20 000 reticulocytes per μl in the peripheral blood, occurred in both series after a similar number of days. The overlapping kinetics of hematopoietic recovery translated into comparable clinical care (Table 3). No relevant side-effects attributable to IL-2 administration were observed. Transfusion requirements were comparable in our series and consisted of a median of one single-donor platelet unit in both groups. Hospital stay did not differ significantly in the two distinct groups and lasted a median of 17 and 18 days in G-CSF/EPO and G-CSF/EPO plus IL-2 series, respectively. Figure 1 shows blood cell counts of all evaluable patients on day +20, +60 and +100 after PBSCT. Comparable WBC, lymphocyte and platelet counts were observed at the different time points, whereas G-CSF/EPO plus IL-2 patients had significantly higher PMN counts on day +20 and +100, showing an average increase in PMN count of 800 cell per μl on day +100.
Kinetics of lymphocyte reconstitution
The T lymphocyte population rapidly recovered following PBSCT, reaching normal counts within 60 days in both groups (Figure 2). A more in-depth evaluation of subset recovery showed, however, that lymphocyte recovery was mostly ascribable to a fast increase in CD8+ lymphocytes (Figure 2), which were over-represented during the entire follow-up period, confirming previous reports.5 CD4/CD8 lymphocyte ratio reflected this trend, still being very low on day +100 (Figure 2). Analysis of the alternative expression of CD45RA and CD45RO isoforms on recovered lymphocytes was used to assess the presence of naive (CD45RA+CD45RO−) and memory (CD45RA+CD45RO+) subsets, respectively. No marked difference in memory T lymphocyte count was observed throughout the whole study monitoring in the two distinct series (Figure 2). Following a similar decline in the early post-PBSCT period, naive T lymphocytes recovered a significantly higher count on day +100 in G-CSF/EPO group, as compared to G-CSF/EPO plus IL-2 patients (Figure 2). Collectively, subset analysis did not reveal any significant differences between groups on day +100 except in naive lymphocyte count.
Finally, a significant increase of CD3−/CD16+/CD56+ NK cell count was observed in the G-CSF/EPO plus IL-2 series on day +20 (P = 0.020). Subsequent analysis showed that the higher NK count did not persist until day +100, when NK were comparable in the two patient series.
NK cell activity
To document NK activity, a typical NK functional assay was performed in both groups at the different time points.18 Gradient isolated mononuclear cells from both patients’ series were assayed for NK activity through a 51Cr assay. As revealed by flow cytometry, Ficoll–Hypaque isolated mononuclear cells contained all lymphoid subsets, including CD3−/CD16+CD56+NK cells. Additionally, the frequency of CD14+ monocytes among isolated mononuclear cells was similar in the distinct patient series at any time point (data not shown). No statistically significant difference was observed at any time point between the two study groups and day +100 NK activity expressed as % of target cell lysis averaged 15 and 20 in G-CSF/EPO and G-CSF/EPO plus IL-2, respectively. Kinetics of NK activity are shown in Figure 2.
At the time of this analysis, nine patients (one OvCa and eight BrCa) out of 14 G-CSF/EPO patients and 12 (three OvCa and nine BrCa) out of 15 G-CSF/EPO plus IL-2 patients are alive without evidence of disease with a median follow-up of 17 months (range 6–58; median 33, for the G-CSF/EPO group; median 16, range 10–21 for the G-CSF/EPO plus IL-2 group). Three patients (BrCa) treated by G-CSF/EPO were lost to follow-up. One early relapse (<12 months) was observed both in the G-CSF/EPO group (BrCa) and in the G-CSF/EPO plus IL-2 group (OvCa). Four late relapses (>12 months) were observed among G-CSF/EPO treated patients (three OvCa and one BrCa) and two in the G-CSF/EPO plus IL-2 group of patients (one OvCa and one BrCa). One G-CSF/EPO-treated patient (OvCa) died of disease progression 32 months from diagnosis.
The use of high-dose chemotherapy with autologous PBSCT for the treatment of high-risk cancer patients has been widely investigated over the past 10 years. Although phase II trials of stem cell transplantation have provided promising leads,19,20 several phase III studies carried out in BrCa failed to demonstrate consistent survival benefits for patients treated by PBSCT.21,22 The reasons which underlie the lack of significant improvement of disease control by PBSCT over conventional treatment in patients with BrCa are still unclear. Moreover, fatal toxicities related to PBSCT are less than 2–3% in solid tumors, indicating a minimal impact on overall survival. Finally, most patients with metastatic BrCa achieve clinical remission following PBSCT, but they experience relapse a few months later, suggesting that minimal residual disease could be responsible for tumor recurrence. Recent reports by ours and other groups support the hypothesis that an enhanced post-PBSCT immune recovery might contribute in tumor control and patient survival. In fact, a favorable impact of prompt lymphocyte recovery on overall and disease-free survival has been observed in lymphomas, myelomas, breast and ovarian cancer following PBSCT.8,23,24 In particular, a randomized comparative trial carried out by our group showed that both post-PBSCT GM-CSF and G-CSF administration was equally effective in promoting myeloid engraftment, but G-CSF resulted in a more significant short- and long-term immune recovery in BrCa and OvCa patients.8 This difference in terms of immune recovery translated into a survival advantage for the G-CSF arm. This last finding supports the hypothesis that tumor control by the immune system may also be operational in patients with epithelial neoplasms. In this context, post-PBSCT administration of IL-2 might enhance immune reactivity against residual tumor cells and potentiate post-transplant lymphopoiesis acting on primitive CD34+CD105+ cells which express high-affinity IL-2 receptor and on which IL-2 produces a lymphoid-oriented differentiating stimulus in vitro.25 In fact, our studies on functional characteristics of circulating CD34+ cells revealed that a CD34+/CD105+ subset expresses considerable levels of CD25 and retains the ability to generate lymphoid precursors and differentiate in vitro along the pre-thymic lymphoid pathway in the presence of a cytokine mixture containing low-dose IL-2.
In the light of this experimental evidence, a prospective study was undertaken to verify whether IL-2 in addition to G-CSF/EPO early after PBPCT may further improve immune recovery and function in patients with high-risk BrCa or advanced OvCa. The primary endpoint of the study was comparison of immuno-hematopoietic reconstitution driven by G-CSF/EPO plus IL-2 and by G-CSF/EPO. A secondary endpoint was evaluation of the short- and long-term effect of low-dose IL-2 on natural killer activity (NK activity) exerted by recovered lymphocytes from transplanted patients.
Collectively, our results showed that immuno-hematopoietic recovery and post-transplantation clinical care were comparable in the G-CSF/EPO- and G-CSF/EPO plus IL-2-treated patients without significant side-effects attributable to IL-2 administration. These observations are similar to those described by Laughlin et al,26 who reported no relevant modifications of marrow regeneration using continous intravenous infusion of similar doses of IL-2 (40 000 Cetus units m2/day equivalent to 240 000 IU m2/day) early after BMT. Similarly, Meehan et al27 observed no modification of engraftment time using higher IL-2 dosages (600 000 IU m2/day) subcutaneously. Our present study extends these findings to patients receiving concomitant IL-2/G-CSF/EPO. In particular, we observed a significantly higher PMN count in the G-CSF/EPO plus IL-2-treated patients in the early and late post-PBSCT period and these findings fit well with a similar enhancement of PMN recovery reported by Heslop et al.28 On the other hand, the effect on PMN recovery was devoid of particular clinical relevance and an insignificant, pre-PBSCT, higher PMN average count in G-CSF/EPO plus IL-2 series must be taken into account as a possible contributor to the better post-PBSCT PMN recovery in these patients. Nonhematological toxicity of the G-CSF/EPO plus IL-2 series remained comparable to that of the G-CSF/EPO group, with no increase in the frequency of gastrointestinal toxicity as previously shown by Toh et al,29 who administered IL-2 at a dose of 500 000 IU/m2/day early after PBSCT in metastatic BrCa. Our interest in evaluating NK activity in our patient groups was prompted by two observations. First, lymphocyte proliferative response, LAK and CTL functions are known to decrease following subcutaneous low-dose IL-2 administration to HIV-infected individuals and NK activity, following an initial increase, decreased below baseline levels during treatment of these patients.30 Second, impaired NK cell activity might adversely impact on the control of minimal residual disease in cancer patients. Thus, an additional finding of this study was that NK activity was not significantly depressed after low-dose IL-2 treatment at any time point. The recently reported enhancement of NK cell function after high-dose i.v. IL-2 administration supports the view that different doses and duration of IL-2 treatment may differentially affect immune parameters.31 However, data from the present study showed a certain dissociation between the absolute number of cells expressing a NK phenotype and relative lytic activity. Particularly, G-CSF/EPO plus IL-2 patients had significantly higher CD3−/CD16+CD56+ counts on day +20 without a parallel enhancement of NK activity. Isolation procedures to obtain cell suspensions containing effector cells for 51Cr assay were identical in the two distinct patient series, so that the observed results suggest that some phenotypically defined NK cells lacked lytic functions on day +20 in G-CSF/EPO plus IL-2 patients. The immune mechanism which determines a defect in NK activity in these patients is unclear, but this finding is in line with the reported ineffectiveness of IL-2 treatment in promoting post-PBSCT NK functions in childhood cancer.32 Alternatively, we can hypothesize that the classical NK functional assay is not so sensitive as to identify a change in functional activity which should derive from the doubling of phenotypically defined NK cells.
An unanticipated finding of the study was the observation of a lower count of naive T cells in the G-CSF/EPO plus IL-2 compared with the G-CSF/EPO group. A similar reduction in CD45RA+ T cell count has already been reported at late time points after subcutaneous low-dose IL-2 administration.30 This phenomenon has no apparent explanation and suggests that, similar to myelopoietic growth factors, IL-2 given soon after PBSCT also produces long-lasting immune effects with persistent modulation (observable until day 100 post-PBSCT) in the number of de novo generated T cells (ie naive CD45RA+ T cells). The detrimental effect of IL-2 addition on CD45RA+ T cell regeneration is surprising and confirms the lack of favorable post-PBSCT immune effects by IL-2. As anticipated above, we observed a similar phenomenon with the administration of the myelopoietic growth factor GM-CSF which transiently enhanced post-PBSCT PMN recovery (as compared to G-CSF) but impaired long-term T cell regeneration.8 The coexistence of enhanced myelopoiesis (as revealed by increased PMN counts) and defects in T cell counts favors the hypothesis that IL-2 and GM-CSF expand post-PBSCT myelopoiesis at the expense of T cell regeneration through a mechanism of in vivo stem cell competition. Additionally, a recent report by Aladdin et al33 showed that subcutaneous IL-2 administration shortens telomere length of peripheral blood mononuclear cells of individuals infected with HIV with selective expansion of memory T cells and concomitant reduction of both CD4+CD45RA+ and CD8+CD45RA+ naive lymphocytes, which is highly reminiscent of our present observations. Finally, the small size of our patient sample, the short follow up of G-CSF/EPO plus IL-2 series and the lack of relevant differences in the rate of early and late relapse between our distinct patient series prevent any preliminary conclusion on the clinical role of post-PBSCT IL-2 addition to G-CSF/EPO regimen. Based on this knowledge, further studies aimed at evaluating T cell function after low-dose IL-2 treatment are needed to test whether decreased naive T cells counts are associated with specific T cell functional defects.34 Results from this comparative study demonstrated that low-dose IL-2 did not modify hematological and non-hematological toxicities of PBSCT in patients receiving concomitant G-CSF/EPO. Moreover, IL-2 administration enhanced the magnitude of PMN recovery confirming the hypothesis that low-dose IL-2 can directly or indirectly affect the differentiating capacity of transplanted PBSC, even though no favorable effects were observed on lymphoid recovery. Finally, low-dose IL-2 did not modify NK activity after PBSCT. Further studies should verify the role of IL-2, in addition to well-established post-PBSCT cytokine regimens on survival and disease control in patients with cancer.
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