Patients and methods

Patients

Between December 1998 and April 2001, 90 patients underwent an RIC allogeneic peripheral blood stem cell transplantation (PBSCT) in nine Spanish institutions within a prospective trial. Details of the study have been previously published.⁷ A total of 70 consecutive patients from four of the participating institutions were included in the current study. Two patients died on days +9 and +22 from pneumonia and multiorgan failure and were not included in the analyses.

Patient characteristics are shown in Table 1. The median age was 52 years (range 19–62). Disease phase at transplant was categorized as early in 17 (25%) patients (acute leukemia or poor-risk myelodysplasia in first complete remission, untreated good risk myelodysplasia, first chronic phase of chronic myeloid leukemia (CML), lymphoid malignancy in first remission), intermediate in 21 (31%) patients (acute leukemia or myelodysplasia in second or higher complete remission, accelerated phase CML or lymphoid malignancy in second or higher remission) and as advanced phase in 30 (44%) patients (refractory or relapsed acute leukemia or myelodysplasia, blastic phase CML and all second transplants).

Table 1 - Patient characteristics.

Full table

Disease status at transplant differed between the two conditioning regimens. In all, 24 (60%) patients with advanced disease were transplanted in the melphalan regimen, whereas only six (21%) patients who received the busulphan regimen were in an advanced status at transplant (P=0.001). Additionally, a prior HSCT had been performed in 19 (49%) and 5 (18%) patients, respectively (P=0.01). Consequently, melphalan recipients were more heavily pretreated than busulphan recipients; the number of patients who had received more than two lines of chemotherapy (including a prior transplant as a 'line of chemotherapy') were 22 (55%) and three (11%), respectively (P<0.001).

Conditioning regimens

Two different RIC regimens were used, one for lymphoid and one for myeloid malignancies except for four patients who were given the alternative regimen since they had received a prior transplant that included in the conditioning regimen the assigned drug (Table 1). The myeloid RIC regimen (n=28) consisted of fludarabine 30 mg/m² intravenously (i.v.) between days -9 and –5 and busulphan 1 mg/kg 10 doses on days –6 to –4 (total 10 mg/kg). The lymphoid regimen (n=40) consisted of fludarabine 30 mg/m² i.v. on days –8 to –4 followed by melphalan 70 mg/m² i.v. on day -3 and -2. PBSC were infused on day 0.

Graft versus host disease (GVHD) prophylaxis and supportive care

GVHD prophylaxis consisted of cyclosporine A (CsA) and a short course of methotrexate. CsA was given at a dose of 1 mg/kg/day i.v. or by mouth from day -7 to -2, and then 2 mg/kg/day from day -1. Levels were maintained in the therapeutic range until tapered. In cases when acute GVHD grade>1 did not develop, CsA was tapered 10% weekly starting on day +90 and discontinued if no GVHD appeared by day +150. Methotrexate was given at a dose of 10 mg/m² on days +1, +3 and +6, followed by folinic acid rescue. Acute and chronic GVHD were graded according to established criteria.⁸ Moderate-to-severe GVHD was defined as the presence of grades II–IV acute GVHD and/or extensive chronic GVHD. Standard supportive care was used in all cases, as specified elsewhere.⁷

Chimerism analysis

Serial samples of peripheral blood (PB) and bone marrow (BM) were analyzed for degrees of donor–recipient chimerism using PCR of informative minisatellite loci. Samples were obtained at least after hematological recovery (days +21 to +30), at 3 months, 6 months, 9 months, 1 year, and between 18 and 24 months post transplant. Additional samples were analyzed if deemed appropriate by the attending physicians. Chimerism studies were performed with a commercially available automated kit (profiler Plus; Perkin-Elmer, Norwalk, CT, USA) with semiautomatic electrophoresis (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The kit uses multiplex amplification of nine STRs loci (D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11 and D18S51) plus the gender marker Amelogenin that allows quantification of the percentage of donor and recipient DNA in the sample studied. DNA was extracted following standard protocols. To evaluate lineage chimerism, lysis buffer was added to PB and a selection of CD3+ve and CD19+ve cells from PB was done using immunomagnetic beads.

Complete donor chimerism (CDC) was defined as the presence of at least 95% donor DNA in the sample analyzed. Definition of CDC was based on the donor chimerism of T cells if available (n=26) or unfractionated (UF) nucleated cells (n=42). Stable CDC was defined as the presence of two consecutive CDC in PB without later graft failure. Early CDC was defined as the establishment of stable CDC in PB samples within the first 100 days after transplant.

Donor lymphocyte infusions (DLIs)

Our protocol recommended DLIs only in case of progressive loss of donor chimerism or disease progression without prior or concurrent moderate-to-severe GVHD. With this criteria DLIs were given to eight patients, all of whom had previously reached stable CDC without GVHD and showed signs of progression of the underlying disease.

Statistical analysis

The ² statistic or Fisher's exact test was used to establish differences in the distribution of discontinuous variables and Student's t-test or Mann–Whitney's U test to compare continuous variables. All reported P values are two sided, and a significance level of 0.05 was used. Time to onset of CDC was calculated from the time of transplantation using Kaplan–Meier product-limit estimates. Patients were censored at the time of DLI, disease progression (if further salvage chemotherapy was given) or death from any cause, and those who were still alive and progression free at the time of reporting were censored at the last follow–up date. The probability of disease progression post-transplant was calculated using cumulative incidence estimate, with transplant-related mortality considered a competing risk. Logistic regression was used for multivariate analysis of the variables that influenced the achievement of early CDC. Variables included in the models were age, underlying disease (myeloid vs lymphoid), conditioning regimen, status at transplant, prior HSCT, number of prior lines of chemotherapy received (2 vs >2) and CD34+ cell/kg infused with the graft. In order to determine whether the achievement of early CDC influenced the risk of suffering disease progression post transplant, multivariate Cox proportional hazards regression analyses were done. Variables included in the models were age, underlying disease (myeloid vs lymphoid), status at transplant, prior HSCT, number of prior lines of chemotherapy received (2 vs >2), GVHD and the onset of CDC. The occurrence of acute GVHD grades II–IV or extensive chronic GVHD and time to onset of CDC were included as time-dependent covariates, and the assumption of proportional hazards over time was tested for all explanatory covariates using a time-dependent covariate.

Results

A total of 433 samples from the 68 evaluable patients were analyzed during the study. Of these, 209 BM samples (202 UF and seven fractionated), and 224 samples of PB were analyzed; UF cells were studied in 108 samples, fractionated cells in 48 samples and both analyses were done in 68 samples. In these latter samples, chimerism analyses of UF cells and fractionated T-cells showed concordant results in 64 cases (94%) (ie: CDC in 63 and mixed chimerism (MC) in one case). In the other four cases, the percentage of donor cells in UF and T-cells were 100/65, 96/67, 97/85 and 100/90.

Chimerism was analyzed in UF nucleated cells from at least one sample of BM or PB in 56 (85%) patients in at least one occasion, while in 42 (62%) cases fractionated T cells and granulocytes were studied in at least one occasion. Only UF samples were studied in 26 (38%) patients, both UF and fractionated samples in 30 (44%) and only fractionated cells in 12 (18%) patients. Of the 42 patients who had T-cell chimerism studied on at least one occasion, only 26 had at least two consecutive PB samples studied with fractionated cells; these 26 patients will be referred to as those 'studied with fractionated T cells'.

Chimerism analysis in UF nucleated cells

All of the patients showed initial donor engraftment and no patient presented primary or secondary graft failure. On the first chimerism analysis performed around day +30, 44 out of 54 patients analyzed (82%) showed CDC in BM and 43 out of 45 (96%) in PB samples. In those cases with MC the percentage of donor cells ranged from 60 to 94% donor cells in BM (n=10) and 86% in PB (n=2). As shown in Table 2a there were no differences between conditioning regimens. Similar rates of CDC in both treatment groups were found on later time points. Beyond 6 months post-transplant, most patients showed CDC in both BM and PB (see Table 2a). The probability of achieving stable CDC in PB within the first 6 months was 70% on day +30, 85% on day +100 and 95% on day +180 (see Figure 1a).

Figure 1.

Probability of achieving stable complete donor chimerism (CDC) in PB within the first 6 months in (a) all 68 patients and (b) according to whether CDC was defined from unfractionated nucleated cells (n=42, solid line) or T cells (n=26, dotted line).

Full figure and legend (33K)

Table 2 - Complete donor chimerism in (a) unfractionated nucleated cells from bone marrow and peripheral blood after reduced-intensity conditioning allogeneic PBSCT (% in parentheses) and (b) fractionated nucleated cells from peripheral blood after reduced-intensity conditioning allogeneic PBSCT (% in parentheses).

Full table

Chimerism analysis in fractionated nucleated cells (granulocytes and T-cells)

A total of 116 samples were analyzed from PB using fractionated nucleated cells. The analysis of chimerism in fractionated cells showed that early post transplant the presence of CDC was more frequent in granulocytes than in early T cells. Among 22 patients studied around day +30, 21 (95%) showed CDC in granulocytes and 14 (64%) in T cells. As shown in Table 2b, all MC occurred in patients conditioned with busulphan, who had 55–70% donor T cells at this early time point (n=8), while all 14 patients conditioned with melphalan showed CDC both in granulocytes and lymphocytes (P<0.001 for T-cell CDC between groups). In the third month post transplant, 23/26 patients (88%) showed CDC in granulocytes and 15/26 (63%) in T-cells. Again, there were no differences in the rates of CDC in granulocytes among melphalan and busulphan recipients (94 vs 80%, respectively), but there were still significant differences in the rates of CDC in T-cells (93 vs 20%, respectively; P<0.01). In the eight busulphan recipients with T-cell MC at this time point, the level of donor chimerism ranged from 66 to 94%. Thereafter, most patients showed CDC in both granulocytes and T-cells with no differences between melphalan and busulphan recipients (see Table 2b). The probability of achieving stable CDC within the first 6 months post transplant was lower when the analyses was performed in fractionated T-cells (n=26) than when the analysis were performed on UF samples: 43 vs 77% on day +30, 60 vs 92% on day +100 and 85 vs 97% on day +180, respectively (P<0.004) (Figure 1b).

Influence of prior treatments on chimerism status

The factors influencing the probability of achieving early stable CDC were studied. Several factors were found to be statistically significant in univariate analysis. These factors were lymphoid vs myeloid malignancy (90 vs 48%, P<0.001), more than two vs one or two prior lines of chemotherapy (96 vs 60%, P=0.001), having received a prior HSCT (92 vs 54%, P=0.01) and melphalan vs busulphan conditioning (90 vs 50%, P<0.001). However, in multivariate analysis the only significant variable was having received more than two lines of chemotherapy pretransplant (P<0.02).

Chimerism analysis and GVHD

Moderate-to-severe GVHD occurred in 35 out of 59 evaluable patients (41%). A total of 68 patients were evaluable for acute GVHD and 57 patients were evaluable for chronic GHVD, of which 29 (43%) and 37 (65%) developed acute and chronic GVHD, respectively. Moderate-to-severe GVHD developed in 28 of 52 (54%) patients who achieved early CDC and in seven out of 17 (41%) with MC (P=0.4). Among patients whose early chimerism was studied using fractionated T-cells, moderate-to-severe GVHD occurred in nine out of 17 (53%) patients with T-cell CDC and five out of 12 (42%) with MC (P=0.7).

Progression-free survival

The median overall follow-up was 12 months (range, 9–24), and 49 (70%) patients were alive at the time of analyses (November 2001). In multivariate analysis, the only variable that influenced the incidence of disease progression post-transplant was the development of chronic extensive GVHD. The incidence of disease progression at 1 year in patients with (n=49) and without (n=19) chronic extensive GVHD was 6 and 30%, respectively (P<0.05) (Figure 2).

Figure 2.

Cumulative incidence of disease progression after reduced-intensity allogeneic PBSCT in patients who did (n=19) or did not (n=49) develop chronic extensive GVHD.

Full figure and legend (19K)

Discussion

Chimerism analysis is mandatory after RIC regimens for allogeneic HSCT, since the kinetics of lymphoid and myeloid engraftment may differ from those seen after traditional myeloablative conditioning. Many different RIC regimens are currently in use; most protocols use fludarabine, but this drug is combined with low-dose total body irradiation,⁹ low-dose cyclophosphamide^2,5,6 or high-dose alkylating agents,^4,7,10,11 as in our protocols. Additionally, antilymphocyte serums such as antithymocyte globulin and CAMPATH are used in many protocols.^3,12 All of these variables may influence the kinetics of donor cell engraftment, although few detailed studies have been published to date. Our results show that stable CDC can be obtained after our RIC regimens, with no graft failures nor need to use DLI for persistent MC. Myeloid engraftment, as indicated by BM chimerism and granulocyte chimerism in PB, rapidly reached a state of CDC in most cases. The engraftment was stable over time, thus indicating that a stable graft-versus-hematopoiesis effect takes place.

The kinetics of T-cell engraftment differed somewhat between busulphan and melphalan recipients. Although there appeared to be no differences in the rates of CDC achieved between both groups when UF PB samples were studied, this probably reflects the inability to detect a small proportion of residual recipient T-cells in PB hidden among the other predominant cell types of donor origin. This was well reflected in the six samples that had T-cell MC, while UF samples showed >95% donor DNA. As shown in Figure 1b, the kinetics of T-cell engraftment lagged a bit behind that of myeloid (as represented by chimerism studies in UF samples) engraftment. This difference was especially relevant in busulphan recipients, as shown in Table 2b. From these results, one tends to conclude that melphalan is more immunosuppressive than busulphan. However, this may not be the case. Melphalan recipients were more immunosuppressed pretransplant because of their underlying lymphoid malignancy and the heavier pretreatment received. In fact, in multivariate analysis the only variable that was associated with the achievement of CDC was having received more than two lines of chemotherapy pre-transplant. Our results agree with those found by Mattson¹¹ using similar high-dose alkylating agent-based regimens. However, Childs et al⁴ found an earlier rate of T-cell engraftment than myeloid engraftment after a fludarabine-high-dose cyclophosphamide-based protocol. On the other hand, RIC regimens that are immunosuppressive but not myelotoxic, such as the fludarabine-low-dose cyclophosphamide protocol reported by Khouri et al² and the fludarabine-low-dose TBI regimen reported by McSweenwy et al,⁹ inevitably lead to earlier T-cell CDC than myeloid donor chimerism. From our results, we recommend performing chimerism analysis on fractionated T-cells and myeloid cells from PB when using a myelosuppressive RIC protocol, in order to avoid missing cases of MC in T-cells. Once a stable T-cell CDC is reached, UF samples can be used for further studies since graft failures are very rare.

The establishment of CDC has been considered a prerequisite for the onset of GVHD and for a GVT effect, both after conventional myeloablative transplants^13,14,15 and RIC allografting.^3,11,16 We were however unable to find a cause–effect correlation between the onset of CDC and GVHD. Once GVHD occurred, however, all patients were later found to be in CDC. Our results agree with those found by Mattson et al.¹¹ Both studies, however, have similar limitations in their design for finding such an association. Our chimerism studies were performed only twice during the first 100 days post transplant. Since the kinetics of donor cell engraftment can change rapidly early after an RIC allograft, it is possible that patients in MC who later developed GVHD eventually switched to a state of CDC before developing GVHD. The fact that all such patients were in CDC after GVHD was diagnosed supports this hypothesis. In the study by Childs et al,⁴ T-cell chimerism was followed very closely after transplant, and an association was found between the development of CDC and onset of GVHD.

Since most of our patients reached a state of stable CDC in both UF and fractionated T-cells by 6 months post transplant, it is not surprising that we did not find any relation between CDC and disease progression post transplant. After a myeloablative HSCT, the GVT effect is strongly associated with GVHD,^17,18 and the only variable that we found to be protective against disease progression after an RIC allograft was the development of moderate-to-severe GVHD. Since we had very few patients who were in stable MC during follow-up, we were unable to analyze the possible occurrence of a GVT effect in such a setting.

In conclusion, a state of CDC is readily obtained within the first 6 months after our RIC protocols. Donor myeloid engraftment occurs rapidly in all cases, while early T-cell CDC is more common in more immunosuppressed hosts. The high rate of CDC achieved explains the high incidence of chronic GVHD seen, which is similar to unmanipulated PBSCT recipients,¹⁹ and the GVT that occurs after these RIC allografts.

References

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