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Vol. 9, 195–200, January 2003
Clinical Cancer Research 195
Lymphocyte Recovery in Advanced Ovarian Cancer Patients after High-Dose Chemotherapy and Peripheral Blood Stem Cell Plus Growth Factor Support: Clinical Implications1 Gabriella Ferrandina, Luca Pierelli, Alessandro Perillo, Sergio Rutella, Manuela Ludovisi, Giuseppe Leone, Salvatore Mancuso, and Giovanni Scambia2 Departments of Gynecology [G. F., S. M., G. S.] and Hematology [L. P., A. P. S. R., M. L., G. L.], Catholic University of Rome, 00168 Rome, Italy
ABSTRACT Purpose: The purpose of this study was to investigate the clinical role of immunological recovery together with selected biological parameters on long-term survival in a series of ovarian cancer administered high-dose chemotherapy with peripheral blood stem cell and growth factor support. Experimental Design: Thirty-eight patients with stages IIIB–IV epithelial ovarian cancer were studied. Lymphocyte immunophenotyping for the identification of CD3(ⴙ), CD4(ⴙ), CD8(ⴙ), and CD3(ⴚ)/CD16(ⴙ)CD56(ⴙ) natural killer T cells and CD19 B cells was performed. Results: Twenty-three patients (60%) had a CD3(ⴙ) cell count <850 cells/l. Multivariate logistic regression showed that tumor grading (2 ⴝ 6.6, P ⴝ 0.010) and type of growth factor (2 ⴝ 4.1, P ⴝ 0.042) retained an independent role in predicting T-cell recovery above the value of 850 cells/l. The 3-year time to progression (TTP) rate was 86% (95% confidence intervals, 70, 102) in cases with high CD3(ⴙ) cell count with respect to a 3-year TTP of 23% (95% confidence intervals, 8, 38) in cases with low CD3(ⴙ) cell count (P ⴝ 0.0026). The absolute number of CD3(ⴙ) cells was shown to be inversely associated with risk of progression (2 ⴝ 4.8; P ⴝ 0.028), as assessed by Cox univariate analysis using CD3(ⴙ) cell count as continuous covariate. In multivariate analysis only residual tumor and status of CD3(ⴙ) cell counts retained an independent association with shorter TTP. Similar results were obtained for overall survival.
Received 3/12/02; revised 7/24/02; accepted 7/31/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Partially supported by the Italian Association for Cancer Research and Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica. 2 To whom requests for reprints should be addressed, at Department of Gynecology, Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy. Phone and Fax: 39-06-35508736.
Conclusions: Long-term immune reconstitution and particularly the recovery of adequate counts of CD3(ⴙ), CD4(ⴙ), and CD8(ⴙ) T cells are independent markers of longer TTP and overall survival in ovarian cancer patients receiving high-dose chemotherapy with peripheral blood stem cell and growth factor support.
INTRODUCTION PBSCT3 represents an effective supportive strategy for rapid reconstitution of hematopoiesis after HDC regimens (1, 2). Efforts are ongoing to set up the best conditions for an earlier and possibly cell lineage selective growth factor-induced hematopoietic cell recovery (3–5). Evidence has been reported that, besides the advantages of earlier hematopoietic reconstitution and easier clinical management, the use of specific growth factors can play a role in improving immunological recovery: in particular, experiences with allogeneic transplant recipients suggest that restoration and/or maintenance of the immune response might be as effective in controlling microscopic/minimal residual tumor cells that escaped chemotherapy (6, 7). In a different clinical setting, we recently reported (5) the results from a randomized comparison between G-CSF and GM-CSF in the hematopoietic and immune recovery in ovarian and breast cancer patients administered intensive, myeloablative cancer chemotherapy with PBSCT. We showed that although hematopoietic recovery and posttransplant clinical management were comparable in G-CSF- versus GM-CSF-treated patients, significantly higher T cell counts could be found in G-CSF patients during the early and late posttransplant follow-up. Moreover, we reported for the first time in human solid tumors that patients achieving high CD3(⫹) cell count at long-term follow-up showed a longer TTP, suggesting that growth factor-driven improvement in immunological recovery could play a role in post-PBSCT control of disease and result in a survival benefit (5). Although a more in-depth analysis of the association between enhanced recovery of T cells in the post-PBSCT period and a more favorable prognosis needs to be carried out, the possibility that biological characteristics of the tumor can also play a role in influencing tumor/host interactions and patient outcome cannot be ruled out. In particular, there is evidence that qualitative and/or quantitative alterations of tumor suppressor genes (such as p53; Ref. 8) and/or oncogenes (like members of the erbB family; Ref. 9) can identify ovarian cancer patients
with a poor chance of response to chemotherapy and poor prognosis. To our knowledge, few data are available addressing the issue of the possible clinical role of biological factors as well as lymphocyte recovery in the peculiar clinical setting constituted by patients with solid tumors administered HDC (10). The aim of this study was to investigate the clinical role of early immunological recovery together with selected biological parameters on long-term survival in a series of advanced ovarian cancer patients with minimal chemosensitive disease, administered HDC with PBSC and growth factor support (11).
PATIENTS AND METHODS Thirty-eight patients with histologically confirmed, advanced (International Federation of Gynecology and Obstetrics stages IIIB–IV) epithelial ovarian cancer, residual tumor ⱕ2 cm achieved at primary cytoreductive surgery, or interval debulking surgery without signs of progression after induction chemotherapy were enrolled into a Phase II study investigating G-CSF versus GM-CSF effects after HDC with PBSC and growth factor support (11). The length of median time to study entry for mobilization chemotherapy was 3 weeks (range, 2– 4; SD, 0.80) and 3 weeks (range, 2– 4; SD, 0.87) for patients submitted to primary debulking surgery or interval debulking surgery, respectively. Other eligibility criteria were: age younger than 60 years; performance status of 0 –1 (WHO scale); adequate bone marrow reserve (WBC count, ⬎4000 ⫻ 106/liter; platelet count, ⬎100 ⫻ 109/liter); and adequate pulmonary, cardiac, hepatic, and renal functions, as described previously (11). Treatment Plan and Supportive Care. Breifly, the induction phase consisted of only one cycle of cisplatin (100 mg/m2), epirubicin (110 mg/m2), and paclitaxel (175 mg/m2), followed by rh-G-CSF (5 g/kg/day) s.c. as PBSC mobilizing treatment (12). Leukaphereses were performed using the Fresenius AS104 blood cell separator (Fresenius, St. Wendel, Germany). A minimum of 4 ⫻ 108 peripheral blood mononuclear cells/kg or 2.5 ⫻ 106/kg CD34(⫹) cells were collected per patient (13). An additional two cycles of the same regimen were administered. The intensification regimen consisted of carboplatin (600 mg/m2, days 1 and 2), etoposide (450 mg/m2, days 1 and 2), and melphalan (50 mg/m2, days 3 and 4). PBSCs were reinfused on day 5. Twenty-four hours later patients received rh-erythropoietin at a dose of 150 IU/kg s.c. every 48 h until day ⫹11, plus 5 g/kg/day rh-G-CSF s.c. until day ⫹12, or rh-GMCSF (5 g/kg/day) s.c. until day ⫹12. In particular, the dose of GM-CSF was selected on the basis of the range of doses commonly used and reported in the literature specifically in setting of autologous transplantation (5, 14, 15). Hematopoietic engraftment was defined as the number of days necessary to reach WBCs ⬎ 1,000 per l, polymorphonuclear leukocytes ⬎500 per l, and platelets ⬎50,000 per l (11). Long-Term Hematological and Immunological Follow-up. As previously reported (5), to evaluate the hematological and immune status during the late posttransplant follow-up, blood cell counts and circulating lymphocyte immunophenotyping were monitored after an interval from PBSC of 12 months in all evaluable patients receiving either G-CSF or GM-CSF.
Circulating lymphocyte immunophenotyping for the identification of CD3(⫹), CD4(⫹), CD8(⫹), and CD3(⫺)/ CD16(⫹)CD56(⫹) NK T cells and CD19 B cells was performed as described previously (5). Immunohistochemistry. For p53 immunohistochemical assessment, the DO7 monoclonal antibody (diluted 1:100; DAKO, Carpinteria, CA) was used. For Her-2/neu assay, we used the high-affinity murine monoclonal antibody 300G9 (Ig2␣; 50 g/ml), recognizing an epitope of the Her-2/neu extracellular domain, which has an 80.3% concordance rate with protein expression (16). EGFR staining was performed by using the monoclonal antibody 108 (used as culture supernatant diluted 1:4) directed to the extracellular domain of the receptor. Immunohistochemical analysis of p53, Her-2/neu, and EGFR was performed as described previously (17, 18). For p53 analysis, cases were scored on the basis of the intensity of staining and the proportion of cells stained, and judged as negative in the absence of any staining and positive in cases of staining in ⬎1% of cells (corresponding to the median value). Scoring for HER-2/neu was assigned according to the intensity of staining and graded from 0, 1⫹, 2⫹, 3⫹. Strong immunohistochemical reaction (3⫹) was considered as Her-2/ neu positivity. EGFR immunostaining was scored on the basis of the fraction of stained tumor cells: negative (fraction of stained cells ⬍20%) or positive (fraction of stained cells ⬎20%). The analysis of all tissue sections was done without any prior knowledge of the clinical parameters or other immunohistochemical results, by two different pathologists by light microscopy. In case of disagreement, consensus was reached by a joint reevaluation using a multi-head microscope. Statistical Analysis. Mann-Whitney nonparametric test was used to analyze the distribution of CD3(⫹) cells according to several variables. The 2 test and Fisher’s exact test for proportion were used to analyze the distribution of clinicopathological parameters according to different patient populations. Multiple logistic analysis (19) was used to analyze the role of clinicopathological parameters as predictors of CD3(⫹) cell recovery. OS and TTP were calculated from the date of diagnosis to the date of death/progression or date last seen. Medians and life tables were computed using the product-limit estimate by the Kaplan and Meier method (20), and the log rank test was used to assess the statistical significance (21). To reduce the possible bias related to the use of an arbitrary cutoff required in the Kaplan and Meier analysis, we also analyzed the prognostic role of CD3(⫹) cell count as a continuous variable, by the Cox Mantel method (22). Statistical analysis was carried out using SOLO (BMDP Statistical Software, Los Angeles, CA). Median follow-up was 38 months (range, 13–107). Analysis was as of December 2001.
RESULTS During the late posttransplant follow-up, erythrocyte, granulocyte, and platelet counts were comparable in G-CSF- versus GM-CSF-treated patients whereas WBC and circulating lymphocytes were significantly higher in the G-CSF series (Table 1). Cytofluorimetric analysis showed that CD3(⫹) (mean ⫾ SE values, 966 ⫾ 91 versus 674 ⫾ 48; P ⫽ 0.028), as well as
Patients’ blood cell counts after a 12-month follow-up in GM-CSF- versus G-CSF-treated patients GM-CSF
WBC (/l) Granulocytes (/l) Lymphocytes (/l) Platelets (/l) a
3,350 2,000 950 190,000
2,000–6,180 1,100–3,600 280–2,000 125,000–355,000
4,500 2,800 1,600 185,000
2,160–7,600 1,350–5,450 550–2,980 110,000–270,000
⬍0.0001 n.s. ⬍0.0001 n.s.
Calculated by Mann-Whitney nonparametric test. n.s., not significant.
Distribution of CD3⫹ cell counts according to clinicopathological characteristics and type of growth factors CD3⫹ cell count (mean ⫾ SE)
All Age (yr) FIGOc stage III IV Grading G1/G2 G3 Residual tumor ⬍2 cm ⬎2 cm Ascites No Yes Histotype Serous Other Growth factor G-CSF GM-CSF
38 23 15
835 ⫾ 59 891 ⫾ 73 750 ⫾ 98
857 ⫾ 62 583 ⫾ 98
1093 ⫾ 116 743 ⫾ 61
Cases with ⬍850 CD3⫹ cells/l n (%)
23 (60) 12 (52) 11 (73)
20 (57) 3 (100)
2 (20) 21 (75)
828 ⫾ 71 857 ⫾ 111
17 (61) 6 (60)
964 ⫾ 108 751 ⫾ 64
7 (47) 16 (69)
850 ⫾ 72 781 ⫾ 77
18 (60) 5 (62)
966 ⫾ 91 674 ⫾ 48
9 (43) 14 (82)
Calculated by Mann-Whitney nonparametric test. b Calculated by Fisher’s exact test for proportion. c FIGO, International Federation of Gynecology and Obstetrics.
CD4(⫹), CD8(⫹), but not CD3(⫺)/CD16(⫹)CD56(⫹) were higher in G-CSF-treated versus GM-CSF-treated patients (data not shown). No difference in the counts of B lymphocytes was observed (data not shown). CD3(⫹) cell counts at 12-month follow-up ranged from 350-1850 cells/l (mean ⫾ SE values, 835 ⫾ 59 cells/l). The cutoff of 850 CD3(⫹) cells/l was chosen a priori without any prior knowledge of the clinical parameters or patient clinical outcome to define patients with low versus high T-cell recovery at the 12-month follow-up period. Twenty-three patients (60%) had a CD3(⫹) cell counts ⬍850 cells/l. There was no difference in the distribution of the absolute CD3⫹ cell counts according to age, stage, residual tumor, ascites, and histotypes (Table 2). However, patients with poorly differentiated tumors showed a lower CD3(⫹) cell count with mean ⫾ SE values of 743 ⫾ 61 cells/l with respect to 1093 ⫾ 116 cells/l in cases with well/moderately differentiated tumors (P ⫽ 0.0059). Similar results were found when considering the percentage of cases with low CD3⫹ cell counts (20% versus 75% in G3 versus G1–2 tumors, respectively, P ⫽ 0.0037). Patients with G1–2 tumors were found to be older than patients with G3 tumors (mean ⫾ SE, 42.1 ⫾ 2.8 versus 49.0 ⫹ 1.2; P ⫽ 0.027).
We failed to find any correlation between the CD3(⫹) cell count and graft characteristics such as the number of PBSC or CD3(⫹) cells infused. Moreover, we did not find any association between the number of lymphocyte count at diagnosis and CD3(⫹) cell recovery at the 12-month posttransplant (r ⫽ 0.1, P ⫽ 0.6). Finally, no correlation between CD3(⫹) cell count and the time from diagnosis to PBSC collection (P ⫽ 0.5) or transplant (P ⫽ 0.5) was found. Twenty-one of 38 (55.2%) cases showed positive immunoreaction for p53, whereas 8 of 38 (21.0%) and 18 of 38 (47.3%) showed positive immunostaining. No association between the status of biological parameters and final CD3(⫹) cell count at the 12-month interval was observed. Interestingly, multivariate logistic regression showed that tumor grading (2 ⫽ 6.6, P ⫽ 0.010) and type of growth factor (2 ⫽ 4.1, P ⫽ 0.042) retained an independent role in predicting T-cell recovery above the value of 850 cells/l. Survival Analysis. During the follow-up period, progression and death from disease occurred in 27 of 38 (71%) and 19 of 38 (50%) cases, respectively. Median TTP was 57 months with a 3-year rate of 86% (95% CI: 70, 102) in cases with high CD3(⫹) cell count with respect to median TTP of 20 months with a 3-year TTP of 23%
Table 3 Univariate and multivariate analysis of clinicopathological parameters and the CD3(⫹) cell count in predicting TTP in ovarian cancer patients Univariate Variable
Fig. 1 A, TTP rate according to the status of CD3(⫹) cell count in ovarian cancer patients. B, plot of the estimates of relative risk of progression as predicted by CD3(⫹) cell counts, calculated by Cox’s hazard regression model.
(95% CI: 8, 38) in cases with low CD3(⫹) cell count (P ⫽ 0.0026; Fig. 1A). The median OS was 107 months with a 3-year rate of 93% (95% CI: 81, 105) in cases with high CD3(⫹) cell count with respect to median OS of 49 months and a the 3-year OS rate of 62% (95% CI: 42, 82; P ⫽ 0.0015; data not shown). The plot of the relative risk of progression of disease as a function of CD3(⫹) cell number is shown in Fig. 1 B. By using CD3(⫹) cell count as continuous covariate, CD3(⫹) cell number was shown to be inversely associated with risk of progression (2 ⫽ 4.8; P ⫽ 0.028) Similar results were obtained when analyzing OS (2 ⫽ 8.5; P ⫽ 0.0036) as assessed by Cox univariate analysis. In univariate analysis large residual disease at first surgery, low CD3(⫹) cell status, stage IV disease, and use of GM-CSF proved to be associated with a higher risk of progression (Table 3). In multivariate analysis only residual tumor and status of CD3(⫹) cell counts retained an independent association with shorter TTP (Table 3). Similar results were obtained when conducting multivariate analysis for OS, even when using CD3(⫹) cell count as a continuous variable (data not shown). Superimposable results in terms of TTP and OS were obtained when patients were dichotomized as having more or less than 400 CD4(⫹)/l or CD8(⫹)/l (data not shown). On the contrary, no difference in the clinical outcome was observed for
Stage III IV Grade 1–2 3 Residual tumor ⬍2 cm ⱖ2 cm CD3⫹ counts High Low Growth factor G-CSF GM-CSF Histotype Serous Other Ascite No Yes p53 Negative Positive EGFR Negative Positive Her2/neu Negative Positive
1 1.34 d
RR1, unadjusted relative risk; RR2, relative risk after adjusting for all the factors listed. b 2 of the model, 14.18; P ⫽ 0.0008. c Only variables with P ⬍ 0.20 in the univariate analysis were included in the multivariate analysis. d Reference category.
patients with more or less than 150 (median value) CD3(⫺)/ CD16(⫹)CD56(⫹) NK cells/l (data not shown).
DISCUSSION This is the first study demonstrating that the absolute CD3(⫹) cell count after the 12-month follow-up after PBSCT is an independent prognostic factor for both TTP and OS in advanced ovarian cancer patients administered HDC with PBSC and growth factor support. Similar results documenting a correlation between the achievement of high lymphocyte count and favorable prognosis have been recently reported in hematological malignancies and metastatic breast cancer (10, 23) and may be related to the well known graft versus tumor effect occurring in allogeneic bone marrow recipients, in which the donor immune system is supposed to contribute to the eradication of residual disease in the host (24, 25). In the autologous setting these results could be explained by a more effective control of residual tumor cells through the action of an increased number of effector T cells, as suggested by our previous reports and by other investigators (5, 10).
It is, therefore, conceivable that the favorable clinical outcome in terms of TTP and OS observed in patients with high CD3(⫹) cell count could be related to a more effective control of residual tumor cells that survived HDC regimens. We recently showed that G-CSF supports a better lymphocyte recovery than GM-CSF in patients receiving HDC, confirming data previously reported in other clinical settings (26). In this context, the availability of growth factors displaying different potential in enhancing T-cell recovery or hematopoietic rescue is of clinical relevance, although a more in-depth analysis of the kinetics of recovery of T subpopulations as well as their functionality is needed. Particularly, the role of specific stem/progenitor subsets present in the graft in determining the speed of CD3(⫹) recovery must be further clarified, even though our analysis and other reports excluded that counts of recovered CD3(⫹) cells are influenced by the dose of CD34(⫹) or CD3(⫹) cells in the graft (23, 27). In the current study, we first showed that besides the use of G-CSF, among clinicopathological parameters tumor grade is significantly associated with higher CD3⫹ cell counts. Whether this association reflects the ability of poorly differentiated ovarian tumors to interfere with growth factor-driven immune cell reconstitution or whether other unknown factors associated with tumor grading could be causally linked to earlier T-cell recovery is a major issue that needs to be addressed considering also that tumor grading failed to be associated with clinical outcome. On the other hand, preexisting (before HDC) low CD3(⫹) counts in patients with G3 tumors may be hypothesized as a tumor-host biological association because of unknown factors that could also contribute to delayed CD3(⫹) recovery after PBSC infusion. Specifically, a contribution of age-related thymic hypoplasia to poor release of thymic emigrants [namely circulating CD3(⫹) cells] could be taken into account (28), because in our series patients with G1/G2 tumors were shown to be significantly older than patients with G3 tumors. It is worth noting that the only study addressing the role of posttransplant T-cell recovery in breast cancer reported more aggressive biological features (prevalence of estrogen/progesterone receptor negativity) in patients with delayed lymphocyte recovery (10). Interestingly enough, the prognostic role of the absolute CD3(⫹) cell counts was retained in multivariate analysis irrespective of the type of growth factor given after PBSC infusion. In the same way, additional statistical analysis revealed that a CD4(⫹) or CD8(⫹) cell predicted a significantly different TTP and OS rate as did the CD3(⫹) cell status, indicating the clinical relevance of adequate counts of both T-cell subsets. Conversely, no differences in TTP and OS were observed according to CD3(⫺)/CD16(⫹)CD56(⫹) NK cells, thus minimizing the role of late NK cell reconstitution. In reference to this, it has been considered that NK cells had a very prompt recovery in most patients and the range of NK cell count at 1 year of posttransplant follow-up was so narrow to prevent the identification of distinct clinical outcome by statistical analysis. All patients in this series received autologous PBSC differently from previous published results in which several sources of progenitor cells were used (12, 23). Therefore, it is conceivable that besides the specific activity of different growth factors, individual repopulating potential or activities of T-cell subsets may influence long-term immune reconstitution. In this
context, the analysis of thymic rearrangement circles of CD3(⫹) cells, which are a useful marker to establish the ontogenic proximity of posttransplant T cells to the thymus (28), could be clinically relevant, as reported in other clinical settings of immune reconstitution (29). Similarly, it is tempting to speculate that the recovery of low-affinity CD3(⫹) cells, which are considered more likely to recognize tumor cells (30), could possibly translate into antitumor-specific cell response. Our data would, therefore, be considered as potential surrogate markers for immunological antitumor response only when a thorough functional characterization of CD3(⫹) cell subtypes recovered after a long time from transplant will be available. In conclusion, we showed that long-term immune reconstitution and particularly the recovery of adequate counts of CD3(⫹), CD4(⫹), and CD8(⫹) cells are independent markers of longer TTP and OS in ovarian cancer patients receiving HDC with PBSC and growth factor support. Indeed, the possibility to predict the repopulating potentiality of each patient on the basis of her own tumor characteristics as well as source, number and subsets of progenitors infused, and mixtures of growth factors, would be clinically relevant to select patients who might benefit from HDC versus patients unlikely to take advantage of intensified regimens, who can be spared the related toxicity. This issue warrants further investigations in a larger series of cases.
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Lymphocyte Recovery in Advanced Ovarian Cancer Patients after High-Dose Chemotherapy and Peripheral Blood Stem Cell Plus Growth Factor Support: Clinical Implications Gabriella Ferrandina, Luca Pierelli, Alessandro Perillo, et al. Clin Cancer Res 2003;9:195-200.
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