Introduction

Despite the introduction of new treatment strategies for chronic myeloid leukemia (CML), hematopoietic cell transplantation (HCT) remains the only curative approach with durable and complete disappearance of BCR-ABL transcripts.^{1, 2, 3} Regimen-related toxicities, however, have limited the procedure to younger and medically fit patients (generally 55 years old). Thus, the majority of patients with CML, given the median age of 65–70 years, are not candidates for conventional HCT.⁴ In an attempt to reduce mortality and make the procedure feasible for older patients, reduced intensity preparative regimens have been developed with the aim to obtain donor cell engraftment and use the graft-versus-tumor (GvT) effect to eradicate the malignant cell clone. Based on preclinical studies, an immunosuppressive rather than myelosuppressive protocol with minimal hematological and nonhematological toxicities was developed. The conditioning consists of TBI 2 Gy pretransplant in combination with CSP and MMF post-transplant to control both engraftment and GvHD.⁵ In a recent study, 45 patients were transplanted from related donors using this regimen.⁶ In a subsequent report, Flu was added to the conditioning in 52 allografts from unrelated donors.⁷ Molecular remissions after related HCT were achieved in five of nine patients with CML, including patients with advanced phase and in four of 12 after unrelated HCT indicating a strong GvT effect. This effect mediated by donor immune cells is modulated by the intensity and duration of the immunosuppressive therapy. Reduction or discontinuation of the immunosuppressive therapy is able to induce complete remissions even in relapsed patients. On the other hand, immunosuppressive therapy after allogeneic HCT controls acute GvHD, which, in contrast to conventional HCT, frequently occurs during tapering or withdrawal of post-transplant immunosuppression.^{6, 7} It is evident that the intensity of immunosuppression must strike the right balance between the risk of relapse and the risk of GvHD. In order to develop a rational approach to this problem, we evaluated variables that might predict the risk of relapse early after transplant. In the present analysis, we demonstrate that a complete clearance of BCR-ABL transcripts is achievable in a significant proportion of patients after HCT following minimal conditioning within 4 weeks from transplant and that the early kinetics of BCR-ABL transcripts significantly correlate with the probability of hematological relapse. Serial measurement of BCR-ABL transcripts may be a useful surrogate marker for monitoring the GvL effect in the early phase after HCT and may open an avenue for early adjustment of immunosuppressive therapy, additional treatment with Imatinib or even more intensive therapies like donor lymphocyte infusion (DLI).

Materials and methods

Between October 1998 and September 2001, 19 consecutive patients with BCR-ABL-positive CML were treated with low-dose irradiation-based regimens at the University of Leipzig. Patients were ineligible for conventional allogeneic HCT because of age (older than 60 years for related and 50 years for unrelated) and/or concomitant diseases or preceding extensive therapies, such as failed autologous or allogeneic HCT. Detailed information on the majority of patients has been published previously.^{6, 7} Characteristics of all patients, disease status and transplant characteristics are given in Table 1. The median patient age was 54 (range 46–63) years. Patients were at the time of transplant in first chronic phase (CP1; n=9), CP2 or CP3 (n=5), accelerated phase (AP; n=4) and blast crisis (BC; n=1). Study protocols and informed consent forms were approved by the Institutional Review Board of the University of Leipzig. Written informed consent was obtained from all patients.

Table 1 - Clinical characteristics and outcomes of 19 patients with CML after related (n=7) and unrelated (n=12) HCT.

Full table

Donors and graft source

Related recipient–donor pairs were completely matched at HLA-A, -B, -C, DRB1 and DQB1 (n=7). Unrelated donors were selected on the basis of serological matching for HLA-A, -B and -C and of allele matching for HLA-DRB1. Either HLA-matched or one antigen mismatched donors were accepted. In addition, all unrelated donor–recipient pairs were retrospectively sequenced and found to be either completely matched (n=7) or 1 mismatched (n=5) at the HLA-A, -B, -C, -DRB1 and -DQB1 allele level (Table 1). Granulocyte-colony-stimulating factor (G-CSF Neupogen^® Amgen, Thousand Oakes, CA, USA) stimulated peripheral blood (PB) cells (G-PBC) were used as source for donor hematopoietic stem cells. The grafts contained a median of 7.6 (range 2.1–24.1) 10⁶ CD34⁺ cells/kg and 4.5 (range 0.2–16.1) 10⁸ CD3⁺ cells/kg.

Treatment

Patients were treated either with Flu 30 mg/m²/day from days –4 to –2 (n=14) or without Flu (n=3) followed by a single fraction of 2 Gy total-body irradiation (TBI) on day 0 delivered at 0.07 Gy/min from linear accelerators. Two patients, who had rejected an unrelated graft after Flu and 2 Gy TBI, received Flu 30 mg/m² on day –4 to –2, Muronomab-CD₃ (Orthoclone OKT3^®) 5 mg i.v. on day –4 to –2, 1 mg/kg i.v., methylprednisone on day –4 to –2 and 4.5 Gy total lymphoid irradiation (TLI) on day 0. CSP was administered orally at 6.25 mg/kg twice daily from day –3. CSP levels were targeted to the upper therapeutic range (500 ng/ml; Abbott TDX, Abbott Park, IL, USA). In the related transplants, CSP was given until day +35 and then discontinued except in patients with GvHD or relapse. Similarly, CSP was tapered after day 100 until day 180 in unrelated transplants. MMF was given orally at 15 mg/kg beginning in the afternoon of day 0 and then twice daily until day +27 while using related donors except in patients with GvHD or relapse. In the remaining patients, MMF was tapered starting on day +40 and withdrawn on day +96 in unrelated transplants correspondingly.

Chimerism analysis

Donor chimerism was detected on sorted CD3⁺, CD56⁺ and CD15⁺ cells either by fluorescence in situ hybridization (FISH) in recipients of grafts from sex-mismatched donors using sex-chromosome-specific probes (SO CEP X/SG CEP Y, Vysis, Stuttgart, Germany) or by polymerase chain reaction (PCR)-based analysis of polymorphic microsatellite regions for recipients of sex-matched transplants.⁸

Definition of relapse and remission

Two consecutive positive tests by conventional two-step nested reverse Transcriptase PCR (RT-PCR) at least 2 weeks apart were classified as molecular relapse and dated from the time of the first detection of BCR-ABL positivity. Accordingly, molecular remissions were defined as two negative results by RT-PCR at least 4 weeks apart. Hematological relapse was defined as described.⁹

Samples, preparation of RNA, c-DNA synthesis, nested RT-PCR and Q-PCR

As per protocol levels of BCR-ABL transcripts in the bone marrow (BM) and PB were measured prior to conditioning and on days +28, +56 and +84 post-transplant. For logistic reasons, assays were performed on days +26.5 (range 22–34), +54 (range 39–62) and +86.6 (range 73–93) after HCT. Samples beyond day +84 were studied routinely by RT-PCR and, in case of positive results, analyzed by quantitative real-time RT-PCR (Q-PCR). Preparation of RNA, c-DNA synthesis, RT-PCR and Q-PCR for BCR-ABL were performed as described previously.^{10, 11, 12, 13, 14}

Statistical analysis

Using the absence or presence of hematological relapse as a dichotomous grouping variable, several potential risk factors were compared either by a nonparametric Mann–Whitney test or ²-test in the case of categorical risk factors. For the purpose of evaluating the effect of day +28 (respectively +56 and +84) BCR-ABL transcript reduction, three univariate logistic regression models, one for each transcript reduction (d+28, +56 and +84, respectively) with relapse as an outcome were used. Based on these models the probability of relapse was estimated and the curves were plotted in one figure to assess consistency of the prediction. Since the study is a cohort-design, the estimated probability of relapse is an estimate of the population probability. No correction for other risk factors was used in order to show the effect of transcript reduction per se.

Results

Comparison of BCR-ABL transcripts in BM or PB

Paired samples from PB and BM were available in 10 out of 19 patients at all time points for a total of 40 PB and 40 BM measurements. No significant differences were observed between the absolute BCR-ABL transcript levels and transcript reductions between PB and BM (data not shown). Further analyses were therefore performed using transcript levels from either PB or BM.

BCR-ABL transcript levels

As shown in Figure 1 transcript levels in BM or PB cells were evaluated prior to conditioning and on days +28 (n=17), +56 (n=16) and +84 (n=15) after HCT. Four measurements (two on day +28 and two on day +84) were missing because of insufficient RNA quality (n=3) and early death (n=1). Three patients had early relapse between day +41 and +57 after transplant. These values were given in brackets in Table 1, but not reported in Figures 1 and 2. Starting from a median of 0.151% (range 0.0185–2.88%) BCR-ABL levels decreased to a median of 0.00604% (range 0.00000167–3.1%) on day +28 after HCT. A further decrease to a median of 0.0000167% (range 0.00000167–0.960%) BCR-ABL transcripts was observed on day +56. Similar ratios were noted on day +84 after HCT with a median of 0.000337% (range 0.00000167–1.54%). The levels, however, showed remarkable fluctuations between patients ranging from undetectable in the RT-PCR to unchanged in comparison to baseline. On day +28 BCR-ABL transcripts were undetectable in six patients by RT-PCR (Figure 1). In all, 11 patients had detectable values by RT-PCR, but ratios by Q-PCR were <0.01% in four and >0.01% in seven patients. On day +56, seven patients were BCR-ABL negative and nine patients BCR-ABL positive by RT-PCR. Of the nine patients with detectable transcripts, seven had ratios <0.01% and two >0.01% by Q-PCR. Finally on day 84, RT-PCR was negative in seven of the 15 patients tested. BCR-ABL transcripts by Q-PCR was <0.01% in six and >0.01% in two patients.

Figure 1.

BCR-ABL transcript levels in 19 patients after reduced intensity HCT. Transcript levels were grouped in high positive (>0.01%) or low positive () (<0.01%). Negative results by nested RT-PCR were shown as (). Values from patients with a subsequent hematological relapse are depicted as black symbols and those from patients with persistent remission as white symbols. Postrelapse values were censored. The difference in BCR-ABL/GAPDH ratio between patients in persistent remission and patients subsequently developing relapse was statistically significant on day 28 (P=0.016 Mann–Whitney test).

Full figure and legend (24K)

Figure 2.

BCR-ABL transcript reduction in 19 patients after HCT with reduced conditioning. Post relapse values were censored. Cumulative transcript reduction among patients with subsequent hematological relapse () and remission () differed at day 28 (P=0.006) and day 56 (P=0.047) post-transplant (Mann–Whitney test).

Full figure and legend (14K)

BCR-ABL transcript reduction

BCR-ABL transcripts on days +28, +56 and +84 were analyzed taking into account BCR-ABL baseline values before HCT (Figure 2). Transcript reductions of more than 4 logs were observed in seven patients within 28 days after HCT. The remaining 10 patients had either no reduction (n=3) or between 0 and <2 log reduction (n=7). On days +56 and +84 after HCT 13 of the 19 patients showed more than 2 log reduction.

BCR-ABL transcripts and relapse

Absolute levels of BCR-ABL/GAPDH as well as reductions in comparison to baseline were correlated with the occurrence of hematological relapse (Figure 1) censoring the postrelapse values in the three patients with early relapse. On day +28, transcript levels of patients remaining in remission were lower than those of patients developing a hematological relapse (P=0.016; Figure 1). All RT-PCR-negative patients remained in hematological remission while only one patient (UPN 709) developed a transient molecular relapse (Figure 3). Of the four patients with low levels on day +28, one (UPN 781) developed hematological relapse and, after discontinuation of immunosuppression, regained molecular remission. One (UPN 730) patient remained in remission with low-level positivity and the other two (UPN 745 and 852) turned RT-PCR negative. Of the seven patients with high levels of BCR-ABL, four relapsed. On day +56, a similar pattern was observed: none of the seven patients with negative BCR-ABL RT-PCR relapsed. Of the seven patients with BCR-ABL <0.01%, only one (UPN 788) relapsed. However, one of the two patients with high levels (>0.01%) had a hematological relapse. On day +84 all of the patients with negative RT-PCR and a low transcript level (<0.01%) remained in remission, while the two patients with high transcript levels relapsed. The differences between patients with and without relapse were even more apparent when BCR-ABL transcript reduction was calculated (P=0.006 on day +28 and P=0.047 on day +56; see Figure 2). On day 28, all seven patients with a reduction of >4 log remained in hematological remission, while four of seven with a reduction of <2 logs and two out of three with no reduction relapsed (Figure 2). On day 56 after HCT, all 13 patients with a reduction of >2 log remained in remission, while only one of the three patients with <2 log reduction remained in continuous remission. On day 84, similar results were seen. None of the patients with more than 2.1 orders of magnitude transcript reduction relapsed. Of the 19 patients, 13 (68%) were alive at 6 months after HCT (Figure 3). Six patients died within 6 months on relapse (UPN 597, 693), GVHD (UPN 730, 889) or infection (UPN 660, 745). At 6 months, five patients were in molecular remission and five patients were either negative by two-step nested RT-PCR at last evaluation or showed low-level transcripts. The remaining three patients had high-level BCR-ABL transcripts. UPN 596 was treated successfully with DLI for hematological relapse on day +56 and reached hematological and later molecular remission on day +300. UPN 781 developed hematological recurrence of CML on day +234 and went back in molecular remission after rapid CSA taper. The third patient (UPN 788) was treated for relapse on day +134 with immunosuppression taper and Imatinib, but died of uncontrolled relapse on day 197.

Figure 3.

Follow-up of BCR-ABL transcripts measured by RT-PCR or Q-PCR in 19 patients after HCT with reduced conditioning. Empty boxes show negative results by nested PCR, gray boxes () illustrate transcript levels below 0.01% but >0 and black boxes () above 0.01%. DLI indicates donor lymphocyte infusion and arrows hematological relapse.

Full figure and legend (61K)

Chimerism and GvHD

As shown in Table 1, all patients engrafted. The chimerism of granulocytes was highest with medians of 95.5% (range 10–100%) and 100% (range 10–100%) donor cells for day +28 and day +56, respectively. Donor T-cell chimerism on day +28 reached a median of 90% (range 27–100%), which subsequently increased to a median of 94% (range 65–100%) on day 56. The donor NK-cell chimerism on day +28 reached a median of 94% (range 26–100%) and increased to a median of 99.7% (range 0–100%) on day +56. Nine out of 19 patients developed GvHD grade 2 until day 84 (Table 1). Five of them had GvHD until day +28, further two until day +56 and another two within +84 days after HCT. GvHD grade 2 did occur in five, six and six out of 14 patients in remission until day +28, +56 and +84, respectively. In comparison, zero, one, and three out of five patients with relapse developed GvHD grade 2 before day +28, +56 and +84 after transplant, respectively (Table 1). In this group, only one out of five patients (20%) had GvHD prior to relapse compared to six out of 14 patients (43%) with remissions (P n.s.). GvHD until day +28 showed a significant correlation with negative RT-PCR results at day 28 (P=0.05) and day 56 (P=0.048), but not at day 84 as determined by the Mann–Whitney test (data not shown).

Additional factors potentially associated with relapse

Various clinical parameters including age, disease stage, gender, unrelated donor, HLA allele disparity, amounts of CD34+, CD3+ and CD56+ cells transplanted, occurrence of GvHD grade 2, donor T-cell, NK and granulocyte chimerism, BCR-ABL transcripts and transcript reduction were analyzed for their association with relapse (Table 2). Factors significantly correlated with relapse were disease stage higher than CP1 (P=0.02), BCR-ABL transcripts on day 28 and transcript reductions on days 28 and 56, respectively. All other factors were not found to correlate with relapse.

Table 2 - Effect of various parameters on the rate of relapse.

Full table

Logistic regression model

To estimate the probability of relapse considering the BCR-ABL transcript reduction at the respective time points, a logistic regression model was created (Figure 4). Analyses revealed an OR=0.30, P=0.07, CI [0.08–1.10] on day +28 and an OR of 0.22 (thus a four-fold reduction in probability) for each unit of magnitude decrease in BCR-ABL expression (P=0.04, 95% CI [0.54–0.9]) on day +56. In contrast an OR of 0.10, P=0.2, CI [0.004–2.4] was calculated on day +84.

Figure 4.

Estimation of the relapse risk by BCR-ABL transcript reduction at different time points after HCT following minimal conditioning using a logistic regression model . Day 28; OR=0.30; CI 0.08–1.095; P=0.07. Day 56; OR=0.22; CI 0.54–0.899; P=0.035. Day 84; OR=0.10; CI 0.004–2.408; P=0.2 (I upper and lower confidence interval (CI).

Full figure and legend (76K)

Discussion

With the advent of reduced intensity conditioning HCT, older patients and patients with concomitant diseases have become candidates for a potentially curative treatment. Several reports investigating feasibility, morbidity and mortality of reduced intensity conditioning protocols in elderly patients with related but also unrelated donors have been published.^{6, 7, 15, 16} Even in patients with mainly immunosuppressive preparative regimens, remissions of the malignant disease were induced by the GvT effect. But GvHD during reduction of immunosuppression in patients with remission of their disease occurred. Therefore, manipulating intensity and duration of post-transplant immunosuppression as critical determinant for GvHD and relapse still remains the key issue in reduced intensity HCT. Immunosuppressive therapy to date, however, has been applied according to a rigid scheme and modified only after relapse or GvHD had occurred. In our analysis, we focused on the possibility to quantify the tumor load after reduced intensity HCT for CML and the predictive value of BCR-ABL transcript determination in comparison to other risk factors for relapse. Several conclusions can be drawn from our study:

First, a complete clearance of transcripts was observed as early as 28 days after HCT in six out of 17 patients. This is clearly faster than reported in a recent study of 15 patients undergoing allogeneic HCT from related or unrelated donors after conditioning with Busulfan, Fludarabine and ATG, and similar to the kinetics of 10 patients after conventional HCT described in the same publication.¹⁷ Interestingly, none of the 15 patients had a negative RT-PCR for BCR-ABL within the first 3 months after HCT, despite the more myelosuppressive preparative regimen. ATG treatment with elimination of host, but also donor immune cells might be responsible for the delayed GvT effect early after HCT. In the same study, five out of 10 (50%) patients after allogeneic HCT with conventional conditioning achieved RT-PCR negativity between days 28 and 84. This is comparable to the seven out of 17 patients (41%) in our study who achieved RT-PCR negativity until day 84. Other authors have assessed BCR-ABL transcripts after conventional conditioning and allogeneic HCT only after day 100 post-transplant.⁹ Intervals to molecular remissions similar to ours were seen after DLI infusions.^{18, 19} Baurmann et al²⁰ reported four out of seven patients achieving molecular remissions after 84 days following DLI.

Second, Q-PCR is an accurate method to measure BCR-ABL transcripts in patients with CML receiving TBI-based regimens. Negative RT-PCR results correlated in 100% of the cases with negative Q-PCR results. Q-PCR, however, was able to further separate patients with low and high transcript levels in patients with positive RT-PCR. The sensitivity and reproducibility observed with our single step real-time Q-PCR assay was similar to results of other groups.^{21, 22, 23, 24, 25, 26} No differences were observed in BCR-ABL transcripts measured by Q-PCR and RT-PCR in BM and PB, confirming previously reported findings.^{27, 28}

Third, BCR-ABL levels and even more the clearance of transcripts early after transplant were consistently associated with hematological remissions. Radich et al^{3, 28} reported RT-PCR negativity between 6 and 18 months after transplant as an important factor for remaining in remission. Low levels of BCR-ABL transcripts were reported to indicate a low risk of relapse compared to high transcript levels and were used as predictive factors in patients after conventional HCT.^{9, 29} Olavarria et al⁹ reported a cumulative risk for disease recurrence of 43% in low-level positive patients and of 86% in patients with high-level residual disease. These findings and others previously published in AML³⁰ were similar to our results. Patients with BCR-ABL levels >0.01% on day 84 relapsed, whereas patients with levels <0.01% remained in remission. BCR-ABL transcript reduction was even better suited to predict hematological relapse, since transcript reductions of >2.1 order of magnitude within 84 days after HCT were consistently associated with hematological remission. Only two out of four patients with a less pronounced transcript reduction remained in remission. To estimate the probability of relapse, a logistic regression model was designed. In this model, a BCR-ABL transcript reduction of more than 2 logs was associated with a probability of relapse of less than 20% and a reduction of more than 4 logs with a probability of relapse of less than 5%. Disease status at transplant was the other factor associated with the occurrence of relapse (Table 2). BCR-ABL determination, however, was predictive for relapse even in patients with advanced disease. Of five patients with CML in AP and BC, four had transcript reductions between +0.6 and -0.6 at day 28 after HCT and subsequently relapsed, while one patient showing a transcript reduction of -5.4 at day +28 remains still in remission more than 30 months after HCT. More than 2.1 transcript reduction was seen in the majority of patients in first chronic phase on day +28 and in all patients on day +56. All patients remained in remission. More than 2.1 transcript reduction was observed also in three of five patients with second or third chronic phase on day +56 without subsequent relapse. Two patients were below 2.1 on day +56, one of them with a subsequent relapse on day +136 and the other without relapse but with GvHD. Despite the restricted number of patients in this analysis, BCR-ABL transcript reduction seems to be predictive independently from disease status and available at the time of MMF taper.

Fourth, no correlation between lineage-specific chimerism and absolute transcript levels or transcript clearance early after HCT was seen. Thus, some patients (UPN 693, 781, 788) had >90% donor chimerism on day 56, but only a limited decrease of BCR-ABL transcripts. This confirms the important role of transcript evaluation, which may reflect the activity of the leukemic clone more accurately than the total amount of remaining leukemic host cells. This parameter was a better predictor of relapse than the percentage of donor cells present in the blood or BM.

Control of GvHD and disease remains a critical future research objective in reduced intensity HCT. Molecular remissions early after HCT appear durable and the logistic regression model of days 28 and 56 may help estimate the risk of relapse. In such patients, immunosuppression taper should focus on GvHD control. Patients positive by RT-PCR must be followed closely by Q-PCR and values might be used to taper immunosuppressive therapy. Additional treatment with Imatinib might be considered.³¹ Protocols have now been designed to prospectively test a tailored immunosuppressive therapy after HCT with minimal preparative regimen.

References

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Acknowledgements

We thank the laboratory and medical staff of the University of Leipzig for their important contribution to this study and especially Ines Kovasc and Scarlet Musiol for their technical expertise in the PCR analysis.