Administration of low-dose interleukin-2 plus G-CSF/EPO early after autologous PBSC transplantation: effects on immune recovery and NK activity in a prospective study in women with breast and ovarian cancer

Administration of low-dose interleukin-2 plus G-CSF/EPO early after autologous PBSC transplantation: effects on immune recovery and NK activity in a prospective study in women with breast and ovarian cancer
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  Bone Marrow Transplantation (2002) 30 , 571–578 ©  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 Immune reconstitutionAdministration of low-dose interleukin-2 plus G-CSF/EPO early afterautologous PBSC transplantation: effects on immune recovery and NKactivity in a prospective study in women with breast and ovariancancer A Perillo 1 , L Pierelli 2 , A Battaglia 1 , MG Salerno 1 , S Rutella 2 , E Cortesi 3 , A Fattorossi 1 ,L De Rosa 4 , F Ferrau` 5 , M Lalle 4 , G Leone 2 , S Mancuso 1 and G Scambia 1 1  Department of Gynaecology and Obstetrics, Catholic University of the Sacred Heart, Rome, Italy;  2  Haematology and  Haemotransfusion Service, Catholic University of the Sacred Heart, Rome, Italy;  3  Department of Experimental Medicine, ‘LaSapienza’ University, Rome, Italy;  4  Haematology, ‘S Camillo-Forlanini’ Hospital, Rome, Italy; and   5  Division of Medical Oncology,‘S Vincenzo’ Hospital, Taormina, Italy Summary:This study evaluated the effects of low-dose IL-2 plusG-CSF/EPO on post-PBSC transplantation (PBSCT)immune-hematopoietic reconstitution and NK activityin patients with breast (BrCa) and ovarian cancer(OvCa). To this end, two consecutive series of patientswere prospectively assigned to distinct post-PBSCTcytokine regimens (from day  + 1 to day  + 12) which con-sisted of G-CSF (5   g/kg/day) plus EPO (150IU/kg/every other day) in 17 patients (13 BrCa and 4OvCa) or G-CSF/EPO plus IL-2 (2    10 5 IU/m 2  /day)in 15 patients (10 BrCa and 5 OvCa). Hematopoieticrecovery and post-transplantation clinical courses werecomparable in G-CSF/EPO- and in G-CSF/EPO plusIL-2-treated patients, without significant side-effectsattributable to IL-2 administration. In the early and latepost-transplant period a significantly higher PMN countwas 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 foundbetween the two groups of patients in the kinetics of most lymphocyte subsets except naive CD45RA + T cellswhich had a delayed recovery in G-CSF/EPO plus IL-2 patients (  P  0.021 on day  + 100). No significant differ-ence was observed between NK activity in the two dif-ferent groups, albeit a significantly higher NK count wasobserved 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 favor-able effects on post-PBSCT myeloid reconstitution, butnot on immune recovery. Correspondence: Dr A Perillo, Department of Gynaecology and Obstetrics,Catholic University of the Sacred Heart, Largo A Gemelli 8, 00168Rome, ItalyReceived 7 January 2002; accepted 28 May 2002  Bone Marrow Transplantation  (2002)  30,  571–578.doi:10.1038/sj.bmt.1703687 Keywords:  IL-2; G-CSF/EPO; PBSCT; immune hemato-poietic recoveryRecent clinical trials have shown a possible role for induc-tion chemotherapy followed by high-dose consolidationtreatment in advanced ovarian cancer (OvCa) patients 1,2 and high-risk breast cancer (BrCa) patients. 3,4 AutologousPBSC transplantation (PBSCT) represents a supportivemeasure to rapidly reconstitute hematopoiesis following theadministration of myeloablative high-dose chemotherapy.Hematopoietic growth factors such as G-CSF or GM-CSFaccelerate neutrophil recovery with a consequent reductionin the number of days with fever and antibiotic treatment,without relevant effects on platelet and erythroid recovery.Theoretically, the clinical use of exogenous cytokines afterPBSCT may cause a selective expansion of hematopoiesisin 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 thatpost-chemotherapy immune-hematopoietic reconstitutionmay contribute to the control of the residual disease. Withreference to this, recent data suggest that reconstitution of immune response could eradicate tumor cells that haveescaped the cytotoxic damage produced by chemotherapy. 5–7 Our previous data showed that both G-CSF and GM-CSFwere equally effective in promoting myeloid engraftmentfollowing PBSCT, but G-CSF promoted a more validimmune recovery. 8 Conceivably, the early administrationof a growth factor involved in T cell development, suchas IL-2, might produce a lymphoid-oriented differentiatingstimulus on transplanted stem cells and activation on  denovo  generated lymphoid cells. To verify the role of post-PBSCT low-dose IL-2 administration, we carried out a pro-spective non-randomized study where G-CSF/EPO-treatedpatients were compared with G-CSF/EPO plus IL-2-treated  Low-dose IL-2 following PBSCT A Perillo  et al  572 Bone Marrow Transplantation patients at several time points from PBSCT. We report theresults of our comparison with respect to hematopoieticrecovery, clinical management, and immune recoveryincluding NK function. Patients and methods Patient’s eligibility and treatment plan Twenty-three patients with resectable high-risk BrCa (stageII–IIIa with   4 involved nodes) and nine patients withstage IIIb-c OvCa with a residual tumor   1 cm after pri-mary cytoreductive surgery or interval debulking surgery(IDS), 9 ranging in age from 33 to 63 years, were enrolledinto this prospective phase II study investigating G-CSF/EPO and G-CSF/EPO plus IL-2 following high-dosechemotherapy with carboplatin (1200 mg/m 2 ), etoposide(900 mg/m 2 ) and melphalan (100 mg/m 2 ) (CEM) andPBSCT. 10 Prior to CEM administration, all patients weretreated with induction nonmyeloablative chemotherapywhich was also used to mobilize and collect PBSC, as pre-viously described. 11,12 None of the patients received radio-therapy. Eligibility criteria included a performance statusof 0–2 (WHO scale), adequate pulmonary, cardiac, hepaticand renal function, absence of underlying infections, aWBC count   2000 per   l and a platelet count   100 000per   l. The study was approved by the Hospital HumanInvestigation Review Board and written informed consentwas obtained from all patients. Patient characteristics at thetime of completion of nonmyeloablative chemotherapy aredetailed in Table 1. Following CEM administration (day  4,   3,   2, and   1) and PBSCT (day 0) the first 17consecutive patients received rhG-CSF (Neupogen; Dom-pe’ Biotec, Milan, Italy) 5   g/kg/day subcutaneously fromday  + 1 to day  + 12. The subsequent 15 patients receivedrhG-CSF 5   g/kg/day subcutaneously from day  + 1 to day + 12, plus concomitant IL-2 (Proleukin; Chiron, Emeryville, Table 1  Patient characteristics, WBC and lymphocyte counts prior toCEM and PBSCT G-CSF/EPO G-CSF/EPO plusn    17 IL-2n    15OvCa (n)  4 5Median age (range) 43 (3850) 42 (3954)Stage IIIb 2 0Stage IIIc 2 5Tumor grade 1/2 1 0Tumor grade 3/4 3 5IDS 1 4  BrCa (n)  13 10Median age (range) 50 (3360) 51 (3663)Stage II 9 8Stage III 4 2Tumor grade 1/2 1 0Tumor grade 3/4 12 10Median of involved 12 (417) 10 (426)nodes (range)CEM    carboplatin, etoposide and melphalan; OvCa    ovarian cancer;BrCa    breast cancer; IDS    interval debulking surgery. CA, USA) 200 000 IU/m 2 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 EPOlevel and to potentiate the effect of G-CSF, as describedpreviously. 11,1315 Oral antibacterial, antifungal and anti-viral prophylaxis was given according to current investi-gational protocols. All patients received a minimum doseof circulating CD34 + cells of 3.0    10 6  /kg body weight.All were transfused with irradiated RBC when the hematoc-rit value was lower than 23% and with irradiated single-donor platelet concentrates when the platelet count waslower than 10 000/   l or during bleeding episodes. Empiricantibacterial therapy was started in the presence of fever  38.5 ° C lasting for more than 12 h and was continued untilthe disappearance of fever for more than 3 days and/orcomplete remission from a clinically or microbiologicallydocumented infectious episode. Hematopoietic recoverywas defined as the number of days necessary to reach 1000WBC per   l of whole blood, 500 PMN per   l, 50 000platelets per   l and 20 000 reticulocytes per   l. FollowingPBPCT, all patients were discharged from the hospitalwhen the PMN count was  500/   l for 3 consecutive daysand platelet count   50 000/   l, in the absence of fever,documented infections or relevant nonhematologicaltoxicities.  Hematological and immunological monitoring Complete blood counts and WBC differentials were takendaily in all patients during their hospital stay, required forCEM administration, PBSC infusion and post-transplan-tation clinical management of hematological and non-hem-atological toxicities. In both patient groups lymphocyteimmunophenotyping with complete blood counts, WBCdifferentials and NK activity were determined on day  + 20, + 60 and  + 100 to document the kinetics of early/latemyeloid/immune recovery. Blood counts were performedfrom samples collected in EDTA using the Bayer Techn-icon H3 RTX System (Bayer Technicon, Tarrytown, NY,USA) and by cytological examination of blood smears forthe assessing of WBC differentials during and after cyto-kine treatment. The variation coefficient (CV) of bloodcounts estimated by the Bayer Technicon H3 RTX Systemwas 3.8% for WBC, 1.4% for hemoglobin and 6% for plate-lets. Reticulocyte counts were performed to documenterythroid recovery by the dedicated and automated reticulo-cyte counter Sysmex R-1000 (Sysmex Toa, Kobe, Japan),as previously described. 16  Lymphocyte immunophenotyping by flow cytometry Immunophenotyping was performed on whole bloodsamples according to the procedures recommended in pub-lished guidelines. 17 Monoclonal antibodies (mAbs; all fromBecton Dickinson, San Jose, CA, USA) with the followingspecificity were used: CD3-, CD4-, CD45-, and CD45RA-fluorescein isothiocyanate (FITC); CD8-, CD14-, CD19-,CD56/16 and CD45RO-phycoerythrin (PE); CD3-peridininchlorophyll protein (PerCP). Dual and three-color fluor-escence was performed using the following combinations  Low-dose IL-2 following PBSCT A Perillo  et al  573 of mAbs: CD14/CD45; CD3/CD56/CD16;CD3/CD45RA/CD45RO; CD3/CD4/CD8; CD3/CD19.Concentration of antibodies was determined by titration andbackground staining assessed by isotype-matched fluor-ochrome-conjugated irrelevant mAbs. After staining, flowcytometric evaluation was performed immediately onunfixed cells on a FACSCan. Absolute counts of the vari-ous lymphocyte subsets were calculated by multiplying thepercentage of cells positively identified by the various mAbcombinations on absolute lymphocyte count. Cytotoxic assay Natural killer (NK) cell activity was assessed using a pre-viously described technique that employs K562 cells as atarget in a 4-h  51 Cr release assay. 18 Several effector/targetratios ranging from 6:1 to 50:1 in replicate wells of a 96-well microtiter plate were established from each mono-nuclear cell sample obtained from patient peripheral bloodcollected at the different time points by gradient isolationusing FicollHypaque (Pharmacia LKB, Uppsala, Sweden;density 1.077 g/ml) and centrifugation at 400  g  for 30 minat 20 ° C. 18 Incubation lasted 4 h at 37 ° C. The specific lysiswas determined according to the previously publishedformula: 18 % of lysis  Experimental c.p.m.  spontaneous c.p.m.Maximal c.p.m.  spontaneous c.p.m.   100%. Spontaneous counts per min (c.p.m.) were determined bymeasuring the amount  51 Cr released from 10 4 target cellsincubated in the absence of effector cells. Maximal c.p.m.were determined by resuspending 10 4 target cells into theassay supernatant. Statistical analysis Comparisons between patient series were performed byMannWhitney  U   nonparametric tests. A  P  value   0.05was considered significant. Results  Myeloid recovery and clinical management followingPBSCT  Seventeen and 15 patients were enrolled in the G-CSF/EPOand G-CSF/EPO plus IL-2 group, respectively, and theircharacteristics at the time of high-dose chemotherapy aredetailed in Table 1. T and B lymphocyte counts prior toPBSCT and the number of CD34  , CD3  and CD19  cellsinfused per kg of recipient weight were similar in the twogroups (data not shown). Following high-dose chemo-therapy and PBSCT, myelosuppression was complete in allcases with a WBC count below 50 per   l and platelet countbelow 10 000 per   l in most cases. All patients were evalu-able for hematopoietic recovery, clinical care and follow-up. The kinetics of hematopoietic recovery, defined as daysto reach 1000 WBC per   l, 500 PMN per   l, 50 000 plate-lets per   l and 20 000 reticulocytes per   l are shown in Bone Marrow Transplantation Table 2. Our results indicate that the number of daysrequired to achieve blood cell recovery was comparable inboth series and similar to that previously described inpatients following CEM and PBSCT. 10 In particular, morethan 500 PMN/   l were reached after a median of 9 daysin both groups, while a platelet count greater than 50 000/   lwas 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, docu-mented by the presence of more than 20 000 reticulocytesper   l in the peripheral blood, occurred in both series aftera similar number of days. The overlapping kinetics of hem-atopoietic recovery translated into comparable clinical care(Table 3). No relevant side-effects attributable to IL-2administration were observed. Transfusion requirementswere comparable in our series and consisted of a medianof one single-donor platelet unit in both groups. Hospitalstay did not differ significantly in the two distinct groupsand lasted a median of 17 and 18 days in G-CSF/EPO andG-CSF/EPO plus IL-2 series, respectively. Figure 1 showsblood cell counts of all evaluable patients on day  + 20,  + 60and  + 100 after PBSCT. Comparable WBC, lymphocyte andplatelet counts were observed at the different time points,whereas G-CSF/EPO plus IL-2 patients had significantlyhigher PMN counts on day  + 20 and  + 100, showing an aver-age increase in PMN count of 800 cell per   l on day  + 100. Kinetics of lymphocyte reconstitution The T lymphocyte population rapidly recovered followingPBSCT, reaching normal counts within 60 days in bothgroups (Figure 2). A more in-depth evaluation of subsetrecovery showed, however, that lymphocyte recovery wasmostly ascribable to a fast increase in CD8 + lymphocytes(Figure 2), which were over-represented during the entirefollow-up period, confirming previous reports. 5 CD4/CD8lymphocyte ratio reflected this trend, still being very lowon day  + 100 (Figure 2). Analysis of the alternativeexpression of CD45RA and CD45RO isoforms on reco-vered lymphocytes was used to assess the presenceof naive (CD45RA  CD45RO  ) and memory(CD45RA  CD45RO  ) subsets, respectively. No markeddifference in memory T lymphocyte count was observedthroughout the whole study monitoring in the two distinctseries (Figure 2). Following a similar decline in the earlypost-PBSCT period, naive T lymphocytes recovered a sig-nificantly 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 significantdifferences between groups on day  + 100 except in naivelymphocyte count.Finally, a significant increase of CD3   /CD16   /CD56  NK cell count was observed in the G-CSF/EPO plus IL-2series on day  + 20 ( P  0.020). Subsequent analysis showedthat 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 assaywas performed in both groups at the different time points. 18  Low-dose IL-2 following PBSCT A Perillo  et al  574 Bone Marrow Transplantation Table 2  Rate of hematopoietic recovery following CEM high-dose chemotherapy and PBSCT G-CSF/EPO n    17 G-CSF/EPO plus IL-2 n    15 P 2  Mean Median Range Mean Median Range Days to:WBC >1000/   l 9.5 9 811 10 9 815 NSPMN >500/   l 9.4 9 811 9.9 9 815 NSPlts >50 000/   l 11.3 12 914 12.2 11 917 NSRet >20 000/   l 11.8 12 1113 11 11 813 NS a Evaluated by MannWhitney  U   nonparametric test.CEM    carboplatin, etoposide and melphalan; Ret    reticulocytes. Table 3  Supportive care, fever and infectious episodes following CEM high-dose chemotherapy and PBSCT G-CSF/EPO n    17 G-CSF/EPO plus IL-2 n    15 P 2  Mean Median Range Mean Median Range Days with fever >38 ° C 1.18 1 04 1.9 2 05 NSDays on antibiotics 1.06 0 07 1.4 0 08 NSClinically documented infections 0 0 — 0 0 — NSMicrobiologically documented infections 0.08 0 01 0.1 0 01 NSRBC transfusions 0.1 0 01 0.2 0 02 NSSingle-donor plts transfusions 1.35 1 03 1 1 02 NSDays in hospital 17.6 17 1621 18.4 18 1523 NS a Evaluated by MannWhitney  U   nonparametric test.CEM    carboplatin, etoposide and melphalan.                                                                                                                      Figure 1  Kinetics of blood cell recovery following carboplatin, etoposide and melphalan (CEM) high-dose chemotherapy and PBSCT in G-CSF/EPO-and G-CSF/EPO plus IL2-treated patients. X axes indicate the days from PBSCT during the post-transplant follow up. Results are expressed as themean    s.d. blood cell counts observed in patients included in each group. *  P    0.034 and  ° P    0.040 at MannWhitney  U   nonparametric test.  Low-dose IL-2 following PBSCT A Perillo  et al  575                                                                                                                                                                                                                                                                                                                                                                                             Figure 2  Kinetics of T cell subset recovery and NK activity (expressed as % of target cell lysis) following carboplatin, etoposide and melphalan (CEM)high-dose chemotherapy and PBSCT in G-CSF/EPO- and G-CSF/EPO plus IL-2-treated patients. X axes indicate the days from PBSCT during the post-transplant follow-up. Results are expressed as the mean    s.d. observed in patients included in each group.  ° P    0.020 and * P    0.021 at MannWhitney  U   nonparametric test. Gradient isolated mononuclear cells from both patients’ ser-ies were assayed for NK activity through a  51 Cr assay. Asrevealed by flow cytometry, FicollHypaque isolatedmononuclear cells contained all lymphoid subsets, includ-ing CD3   /CD16  CD56  NK cells. Additionally, the fre-quency of CD14  monocytes among isolated mononuclearcells was similar in the distinct patient series at any timepoint (data not shown). No statistically significant differ-ence was observed at any time point between the two studygroups and day  + 100 NK activity expressed as % of targetcell lysis averaged 15 and 20 in G-CSF/EPO and G-CSF/EPO plus IL-2, respectively. Kinetics of NK activityare shown in Figure 2. Patients outcome At the time of this analysis, nine patients (one OvCa andeight BrCa) out of 14 G-CSF/EPO patients and 12 (threeOvCa and nine BrCa) out of 15 G-CSF/EPO plus IL-2patients are alive without evidence of disease with a medianfollow-up of 17 months (range 658; median 33, for theG-CSF/EPO group; median 16, range 1021 for the G-CSF/EPO plus IL-2 group). Three patients (BrCa) treatedby 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) andtwo in the G-CSF/EPO plus IL-2 group of patients (one Bone Marrow Transplantation OvCa and one BrCa). One G-CSF/EPO-treated patient(OvCa) died of disease progression 32 months fromdiagnosis. Discussion The use of high-dose chemotherapy with autologousPBSCT for the treatment of high-risk cancer patients hasbeen widely investigated over the past 10 years. Althoughphase II trials of stem cell transplantation have providedpromising leads, 19,20 several phase III studies carried out inBrCa failed to demonstrate consistent survival benefits forpatients treated by PBSCT. 21,22 The reasons which underliethe lack of significant improvement of disease control byPBSCT over conventional treatment in patients with BrCaare still unclear. Moreover, fatal toxicities related toPBSCT are less than 23% in solid tumors, indicating aminimal impact on overall survival. Finally, most patientswith metastatic BrCa achieve clinical remission followingPBSCT, but they experience relapse a few months later,suggesting that minimal residual disease could be respon-sible for tumor recurrence. Recent reports by ours and othergroups support the hypothesis that an enhanced post-PBSCT immune recovery might contribute in tumor controland patient survival. In fact, a favorable impact of promptlymphocyte recovery on overall and disease-free survivalhas been observed in lymphomas, myelomas, breast andovarian cancer following PBSCT. 8,23,24 In particular, a ran-
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