Repeated high-dose chemotherapy (HDC) with stem cell support is advocated for curative treatment of epithelial ovarian cancer patients, requiring large quantities of progenitor cell harvest. Although the switchover to peripheral blood stem cell transplantation has generally made possible the harvest of large quantities of progenitor cells, the minimum threshold is still pertinent for planning the safe conduct of HDC. However, as the minimum threshold for safe peripheral blood stem cell transplantation (PBSCT) is not yet established, this study was designed to clarify the minimum amount of progenitor cells required for prompt recovery of hematopoietic. Retrospective analysis was performed on 52 HDCs administered in 37 ovarian cancer patients. After autologous bone marrow aspiration (10 patients) or peripheral blood stem cell harvest (27 patients), colony-forming unit granulocyte macrophage (CFU-GM) were enumerated prior to cryopreservation. Numbers of CFU-GM were again calculated before reinfusion and the patients were divided into eight groups: 0.13–<0.4, 0.4–<0.7, 0.7–<1.0, 1.0–<3.5, 3.5–<5.0, 5.0–<10.0, 10.0–<20.0 and >20.0 (× 105/kg) . The minimum CFU-GM threshold (× 105/kg) was found to be 1.0–<3.5 for platelets and 3.5–<5.0 for white blood cells. Higher infusion doses did not lead to significant benefits in hematopoietic reconstruction. These results indicate that preservation of a minimum of 7–10 × 105/kg CFU-GM is recommended for the safe conduct of tandem HDCs.
In September 1984, we initiated a trial of high-dose chemotherapy (HDC) with autologous bone marrow transplantation (ABMT) in patients with epithelial ovarian cancer, which was switched to peripheral blood stem cell transplantation (PBSCT) in March 1993. Up to March 1997, 105 patients were given 175 courses of HDC followed by hematopoietic stem cell transplantation following reduction surgery to remove as much tumor as possible. As a result, we have obtained good long-term results, and statistically earlier hematopoietic recovery has been identified in the group given PBSCT.1,2,3,4,5
Recently, several papers have reported the necessity of applying repeated HDCs for curative treatment of solid tumor malignancies.6,7,8,9,10 Mobilization of large quantities of progenitor cells is required for application of several HDCs. Numerous papers have reported on the relationship between the amount of transplanted progenitor cells and hematopoietic recovery. The main factor predicting time to engraftment of autologous blood stem cells is the amount of progenitor cells infused. Several papers report a requirement of more than 2 × 106/kg of body weight,11 5 × 106/kg of body weight12,13 and 15 × 106/kg of body weight14 CD34 cells for shortening time to hematopoietic engraftment and performing HDC with safety. It is necessary to determine not only minimal progenitor cell requirements for recovery of bone marrow function, but also minimum amounts for prompt recovery enabling safe conduct of HDCs. However, most papers do not discuss a maximum PBSCT dose beyond which no significant increase in benefits is obtained. For analyzing this perspective, retrospective analysis was performed to clarify the relationship between the amount of transplanted progenitor cells and recovery of bone marrow function. Apart from the amount of progenitor cells infused, prior chemotherapy regimens, prior radiation therapy and advanced age have been identified as factors having adverse impact on hematopoietic recovery.15,16,17
This study is an analysis of 52 HDCs performed in 37 patients (median age 49.5 years old) with epithelial ovarian cancer, receiving no previous treatment and given the same treatment and HDC described herein.
Patients and methods
Patient selection and HDC regimen
In all, 37 patients (52 HDCs) were selected from 105 patients (175 HDCs) with epithelial ovarian cancer who had received HDCs between January 1994 and December 1997, at the Gynecologic Department of Tokai University. Among the 105 cases, 58 cases were treated with an HDC regimen of cisplatin+adriamycin+cyclophosphamide, and 47 cases with carboplatin+cyclophosphamide. Of these latter 47 cases, the study was conducted on the 37 cases receiving fairly comparable doses – a total of 52 courses of HDC consisting of either 1200 mg/m2 (eight courses) or 1500 (44 courses) mg/m2 of body surface carboplatin (CBDCA)+3000 mg/m2 cyclophosphamide (CPA).
Transplanted progenitor cell counts were 0.13 × 105/kg to 70.55 × 105/kg, with a mean of 11.89±14.32 × 105/kg of colony-forming unit granulocyte macrophage (CFU-GM). Among the 52 HDCs, 16 (10 patients) were given ABMT and 36 (27 patients) PBSCT.
HDC was carried out according to the principles set out in the 1975 Helsinki Declaration and its subsequent amendments, with approval of the Tokai University Review Board. Informed consent was obtained in writing from all patients. Patient characteristics are listed in Table 1. There were no transplant/HDC-related deaths.
ABMT or PBSCT was performed on day 6, and i.v. administration of 300 μg/m2 filgrastim (granulocyte colony-stimulating factor, G-CSF) was continued from day 7 until white blood cell (WBC) counts exceeded 10 000/μl. No statistical difference was noted in hematopoietic recovery between the two regimens with ABMT (Table 2).
Bone marrow aspiration and peripheral blood stem cell harvest
Autologous bone marrow harvest (ABMH) was performed as follows. Between 800 and 1000 ml whole blood was aspirated from the bilateral posterior iliac crest under lumbar anesthesia and subsequently cryopreserved at −190°C according to the method described by Gilmore et al.18 Absence of tumor cells in aspirated bone marrow was confirmed histologically.
Induction chemotherapy for peripheral blood stem cell harvest (PBSCH) consisted of a 1-day administration of cisplatin (50–75 mg/m2)+THP-adriamycin (50 mg/m2) +CPA (750–1500 mg/m2), a regimen also having therapeutic properties for ovarian cancers. One or two courses of this regimen were administered as priming for collection of PBSCs. Daily subcutaneous injection of 50–75 μg/body weight of G-CSF was started from the day on which nadir of leukocytes was observed. After induction chemotherapy, a series of two or three daily leukaphereses were performed using COBE Spectra timed on recovery phase from myelosuppression up to a leukocyte count of 7500/μl.
Enumeration of granulocyte macrophage
The number of progenitor cells collected and transplanted are described in Table 3. Enumeration of CFU-GM was performed by colony assay using commercial colony culture kits19 (Methocult, GF H 4534 Stem Cell Technologies, Inc.) containing stem cell factor, interleukin 3 and GM-CSF. In our institution, CD34+ antigen cells were enumerated when the progenitor cells were collected but not when they were transplanted, as assays post-cryopreservation hold the possibility of including denatured or otherwise nonviable cells in the count. The enumeration of stem cells by CFU-GM assay both at the time of collection and upon reinfusion following HDC was considered an acceptable alternative, given results from a preliminary study of 15 cases that revealed a strong correlation of 0.84 between CD34 and CFU-GM (Figure 1), indicating 1.0 × 105/kg CFU-GM as being comparable to 0.73 × 106/kg CD34 cells. Regarding post-cryopreservation cell loss, comparison of CFU-GM numbers before and after cryopreservation (immediately before transplant) revealed a mean recovery rate of 85.2% (range 67.6–108.2%), or a loss rate of 14.8% (Table 4). From these results, analysis in this study has been carried out using CFU-GM values upon reinfusion.
All patients received hyperalimentation via a central venous catheter and were managed in isolated private rooms or laminar airflow bacterial clean rooms. Irradiated (15 Gy) platelets (PLT) were transfused to keep the PLT count >20 000/μl. Intravenous administration of G-CSF (300 μg/m2) was continued until documentation of bone marrow recovery to 1.0 × 104/μl WBC.
Regarding statistical analysis, a statistician was consulted, and SPSS software was used. Days required for reconstitution of WBC and PLT were regarded as indices of bone marrow function recovery, which are expressed in the bar graphs of Figure 2. Analysis between groups was performed by the Kruskal–Wallis test working with pairs of all groups, and probability was determined to be significant at levels under 0.05. Subsequently, difference between groups was tested by the Mann–Whitney U-test. Data from first and second transplants were treated equally, as analysis of hemopoietic recovery following first and second transplants in the 15 patients receiving second transplants along five parameters (no. of days WBC <1000/μl, day WBC >1000/μl, no. of days WBC <3000/μl, day PLT >3 × 104/μl, no. of PLT transfusions) revealed no significant difference.
There is a distinct difference in the number of autologous progenitor cells collected by ABMH and PBSCH. Compared to ABMH, 17 times as many CFU-GM were collected by PBSCH, making it possible to transplant 21 times as many CFU-GM by PBSCT (Table 3) inducing statistically earlier hematopoietic recovery in the PBSCT group (Table 5). Table 5 shows hematopoietic recovery according to the amount of CFU-GM transplanted by ABMT or PBSCT. Although hematopoietic recovery was obtained in the group receiving under 0.4 × 105/kg CFU-GM, hematopoietic reconstruction was delayed. There was no clear difference between the amount of transplanted CFU-GM and number of days of WBC counts below 100/μl. However, a negative correlation was noted between the amount of CFU-GM transplanted and: (1) the number of days over which WBC counts were <1000/μl or <3000/μl; (2) days required for restoration of WBC counts to 1000/μl or 3000/μl after ABMT or PBSCT; and (3) restoration of PLT counts to 30 000/μl or 50 000/μl (Table 5).
Figure 2 shows a comparison of hematopoietic recovery according to the number of CFU-GM transplanted. As for the recovery of WBC, statistically clear differences were noted between groups receiving CFU-GM transplants under 0.7–<1.0 × 105/kg and over 3.5–<5.0 × 105/kg (Figure 2a and b; P<0.001), or between groups receiving under 1.0–<3.5 × 105/kg and over 3.5–5.0 × 105/kg (Figure 2c; P<0.05). However, transplantation of CFU-GM in quantities exceeding 3.5–<5.0 × 105/kg was not capable of bringing about even earlier recovery of WBC (Figure 2a–c).
As for the recovery in PLT counts, statistically earlier recovery was obtained in groups transplanted in excess of 1.0–<3.5 × 105/kg compared to groups given less than 0.7–1.0 × 105/kg. Although clear reduction was noted in the number of PLT transfusions required (P<0.01) in groups transplanted more than 1.0–3.5 × 105/kg of CFU-GM (Figure 2f), here again, statistical difference was not seen in PLT recovery among the groups reinfused more than 1.0–<3.5 × 105/kg CFU-GM (Figure 2d–f).
The usefulness of HDC with stem cell support against epithelial ovarian cancer has been validated through several retrospective analyses demonstrating high response rates and good long-term results.4,5,20,21,22 However, these results were obtained in selected patient populations characterized by platinum sensitivity, small volume residual tumor and good performance status at transplant. As such, prospective randomized study was required for drawing definitive conclusions on the usefulness of HDC in ovarian caner.23
In 2001, the European Group24 proved, for the first time, the effectiveness of HDC with stem cell support in ovarian cancer by a phase III prospective randomized trial between an HDC with PBSCT group and a conventional dose chemotherapy group in patients with low tumor burden and responsiveness to platinum-based first-line chemotherapy. In addition, several papers6,7,8,9,10 reported that sequential HDC with stem cell support produces high clinical response rates in ovarian cancer. These results uphold the use of multicycle HDC for consolidation after achieving maximal response to conventional chemotherapy, which consequently requires large quantities of progenitor cells. This then necessitates clarification of the minimum threshold dose of progenitor cells for prompt recovery of bone marrow function.
In our analysis of 37 patients receiving 52 courses of HDCs with ABMT or PBSCT, the PBSCT group received reinfusion of 21 times as much CFU-GM, resulting in statistically earlier recovery of bone marrow function (Table 5).
Other papers also report the transplantation of six times25 and seven times26 as much CFU-GM in PBSCT groups, resulting in more rapid hematopoietic reconstruction. Several papers note that as little as 1–2 × 106/kg CD34+ cells or 1–2 × 105/kg CFU-GM27,28,29 were needed for reconstruction of the hematopoietic system, while for rapid neutrophil and PLT recovery, more than 5 × 105/kg CFU-GM30 or 5 × 106/kg CD34+ cells were required. Ketterer et al14 compared neutrophil and PLT recovery in three groups defined by the number of CD34+ cells reinfused: <2.5 × 106/kg (low group), >15 × 106/kg (high group) and an intermediate group receiving reinfusion doses between these two. Their results were that reinfusion of greater than 15 × 106 CD34+ cells/kg after HDC further shortens hematopoietic reconstruction and reduces PLT requirements in addition to improving the patients' quality of life. Perez-Simon et al31 found that although reinfusion of as low as 0.75 × 106/kg CD34+ cells was capable of assuring early engraftment, a dose higher than 1.1 × 106 /kg was desirable in order to avoid transitory late loss requiring PLT and red blood cell transfusions throughout the first year, and that up to a level of 2.2 and 2.4 × 106/kg CD34+ cells, the higher the dose of CD34+ cells infused, the lower the requirement for subsequent red blood cell and PLT transfusion, respectively.32
Lowenthal et al17 showed that, for both granulocyte and PLT recovery, patients receiving ⩾1.89 × 105/kg CFU-GM (median) or ⩾8.8 × 106/kg (median) CD34+ cells showed statistically speedier recovery than patients given <1.89 × 105/kg CFU-GM or <8.8 × 106/kg CD34+ cells.
Our results showed that hematopoietic reconstruction was obtained even in the group receiving less than 0.4 × 105/kg CFU-GM, although this resulted in delayed recovery of WBC and PLT counts in the peripheral blood (Table 6, Figure 2). Groups receiving less than 1.0 × 105/kg CFU-GM showed statistically delayed hematopoietic reconstruction in comparison to other groups receiving ⩾3.5 × 105/kg CFU-GM (Figure 2). However, no difference was seen in WBC recovery among the four groups reinfused with more than 3.5–<5.0 × 105/kg CFU-GM. These results indicate the CFU-GM threshold for prompt neutrophilic recovery as being 1.0–<3.5 × 105/kg CFU-GM (Figure 2a–c). As for PLT recovery, the groups transplanted with less than 0.7–<1.0 × 105/kg CFU-GM showed statistically delayed PLT engraftment (Figure 2d–f) compared to other groups receiving reinfusions of more than 1.0–<3.5 × 105/kg CFU-GM, while no significant difference was observed among the five groups given more than 1.0–<3.5 × 105/kg CFU-GM.
Perez-Simon et al32 report the ideal threshold for infusion to be around 2.2 × 106/kg CD34+ cells, and that infusion over this dose does not lead to a significant increase in benefits after PBSCT. The CD34+ population includes not only CFU-GM but other in vitro colony-forming cells.33,34 Conversion of Perez-Simon's 2.2 × 106/kg CD34+ cells into numbers of CFU-GM according to our calculation based on findings from 15 patients (54 instances), in whom enumeration of both CD34+ cells and CFU-GM revealed high correlation between the two, yields a value of 3.01 × 105 CFU-GM. Being quite similar to our results, we believe that this agreement may be taken as indication of the validity of our evaluation.
From our findings, the following conclusions were drawn. Transplantation of 3.5–<5.0 × 105/kg CFU-GM for neutrophilic reconstruction, and of 1.0–3.5 × 105/kg CFU-GM for PLT reconstruction appears to be both adequate and optimal, as transplantation of CFU-GM over these doses induces no additional benefits in terms of hematopoietic reconstruction. Therefore, harvest of 7–10 × 105/kg CFU-GM, or converting to CD34+ cells, 5.1–7.3 × 106/kg CD34+ cells, is recommended for the safe performance of tandem HDCs, capable of circumventing delayed hematopoietic reconstruction incurring long hospitalizations, improving the patients' quality of life, and with the additional benefit of contributing to significant reductions in costs.
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Cite this article
Miyamoto, T., Shinozuka, T., Maeda, H. et al. Effect of peripheral blood progenitor cell dose on hematopoietic recovery: identification of minimal progenitor cell requirements for rapid engraftment. Bone Marrow Transplant 33, 589–595 (2004). https://doi.org/10.1038/sj.bmt.1704412
- multicycle HDC
- minimum threshold of CFU-GM
- hematopoietic reconstruction
- CD34+ cell
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