In all, 17 consecutive patients in hematological complete remission (HCR) of acute promyelocytic leukemia (APL) received allogeneic stem cell transplantation (SCT) from an HLA-identical sibling and were monitored by reverse transcriptase polymerase chain reaction of PML/RARα prior and after transplant. Median age was 31 years (range 3–50 years). At 10 years, the actuarial probabilities of nonrelapse mortality, relapse and disease-free survival were 32% (95% CI: 8–56%), 33% (95% CI: 6–60%) and 46% (95% CI: 22–70%). Six patients tested PCR +ve (1st HCR n=2; 2nd HCR n=3; 3rd HCR n=1) and 11 PCR −ve (2nd HCR n=11) pre-SCT. Of the six patients PCR +ve, two showed early persistence of PCR positivity and converted to sustained PCR negativity after CSA withdrawal (one died of secondary tumor in molecular remission and one is alive in relapse), while four converted to PCR −ve rapidly (one died of the underlying disease and three are in molecular remission). Of the 11 patients PCR −ve pre-SCT, six died (four of transplant-related mortality, one of relapse and one after heart transplantation) and five are alive, four in molecular remission and one is in relapse. Allogeneic SCT seems a valid option for advanced APL, particularly for the poor prognostic group of patients with pre-SCT molecularly persistent disease.
The outcome of patients with acute promyelocytic leukemia (APL) has dramatically improved following the use of all-trans retinoic acid (ATRA) in association with chemotherapy. 1 As reported in several multicenter studies, up to 70% of newly diagnosed patients receiving this combined treatment are projected disease-free after several years and considered as potentially cured.2,3,4,5,6,7,8,9,10 The results of prospective minimal residual disease (MRD) evaluations conducted in some of these trials indicated that prolonged survival strongly correlates with clearance of the disease-specific molecular marker PML/RARα from patients' blood and marrow cells, at least below the detection threshold of reverse transcriptase polymerase chain reaction (RT-PCR) assays with sensitivity of 10−3/10−4. Conversely, persistence or reappearance of the hybrid transcript after consolidation have been almost invariably associated with subsequent hematologic relapse.3,7,8,10,11 These results led to identify the status of molecular remission as a more advanced and appropriate therapeutic goal for this leukemia and, in some institutions, to adopt the policy of administering additional or anticipated salvage therapy for persistent or recurrent molecular disease, respectively.12
Based on the favorable results obtained frontline with ATRA and chemotherapy, the use of allogeneic stem cell transplantation (SCT) is no longer recommended for APL patients in first hematologic complete remission (HCR1), being the procedure usually postponed to more advanced disease phases, that is, to consolidate ⩾HCR2, or indicated for the rare cases showing persistent MRD after completion of front-line therapy.13 However, only few studies on SCT in APL have been reported to date, and the evaluation of the true antileukemic effect of SCT is hampered in the published series by frequent lack of information on patient pretransplant PCR status.13,14
To gain further insights on the role of SCT in APL, we analyzed our single center experience on 17 consecutive patients who received allogeneic SCT from an HLA-identical sibling in ⩾2 HCR or with molecularly persistent disease and whose pre- and post-transplant marrow PCR status in all cases was prospectively assessed. Our results indicate that SCT may be effective in advanced APL, and suggest that the antileukemic activity of the procedure is mediated by a graft-versus-leukemia (GVL) effect.
Materials and methods
Pretransplant and transplant characteristics
In all, 17 consecutive patients (median age 31 years, range 3–50 years) with genetically confirmed APL in HCR received allogeneic SCT from an HLA-identical sibling at the Department of Cellular Biotechnologies and Hematology of the University La Sapienza of Rome over the period December 1987–July 2001.
Main patient characteristics at presentation, treatment(s) received prior to transplant, time interval between initial diagnosis and SCT, hematologic and PCR status pre-SCT and transplant characteristics are reported in Table 1. As to previous therapies, these included ATRA in all but one patient (#9). In particular, 16 patients had received ATRA plus chemotherapy regimens including in most cases the AIDA protocol of the Italian GIMEMA group.3 All but one patient (#1) received at least two lines of therapy. Three patients (#6, 13 and 17) received autologous transplantation in addition to ATRA and chemo-therapy. All patients were in HCR at SCT, two in HCR1, 14 in HCR2 and one in HCR3. Six patients tested PCR +ve and 11 PCR −ve pre-SCT. All patients received myeloablative conditioning regimens and graft-versus-host-disease (GVHD) prophylaxis based on cyclosporin A (CSA), which was associated with a short course of methotrexate (MTX) in 11. One patient received T-cell-depleted marrow in addition to CSA (#9). Acute and chronic GVHD was diagnosed and graded according to standardized criteria.15,16
Prospective RT-PCR of PML/RARα
Bone marrow (BM) aspirates were obtained at diagnosis, and at regular pre-established time intervals during hematologic remission. In all cases, pre-SCT marrows collected immediately before conditioning regimen were available for analysis. Post-SCT marrows were obtained at 30–90, 120, 150 and 180 days after transplant and then at 6-month intervals until the end of the 5th year post-SCT. Following isolation of the mononuclear cell fraction by centrifugation on a Ficoll–Hypaque gradient, cells were washed twice in phosphate-buffered saline (PBS), suspended in a 4 M guanidium isothiocyanate solution and total RNA was extracted by the method of Chomczynsky and Sacchi.17 Prior to RT-PCR analysis, the integrity of RNAs was assessed by running samples on a formaldehyde minigel. Cases showing partial or total RNA degradation were not processed further and a new sample was requested. Extracted RNAs were reverse-transcribed using random hexamers as primers, following the method described elsewhere.18 The protocol and the oligoprimers used for RT-PCR of the PML-RARα hybrid gene have been reported.3 Amplification of the PML-RARα hybrid gene was carried out simultaneously with the amplification of a RARα cDNA fragment including exon 2 and exon 3 sequences (internal control), in order to further assess RNA integrity as well as the efficiency of the RT step. In addition to hybrid gene and internal control gene amplification, each experiment included a negative control (all reagents plus water) and, as positive controls, RNA from the NB4 cell line or RNA from a BCR3 PML/RARα +ve patient. Serial dilution experiments to assess the sensitivity of the method were carried out periodically, as reported elsewhere.11 A 10−4 dilution of the hybrid gene RNA was consistently amplified in these experiments.
Table 1 shows the outcome and clinical follow-up of individual patients.
Of the eight deaths, two were due to disease relapse occurring at 8 (#7) and 15 (#2) months after transplant, and six were nonrelapse related. The early transplant-related mortality (TRM) was due to acute GVHD in two patients (#11 and 13), primary heart failure in one (#8) and multiorgan failure in one (#12). Finally, one patient (#9) died at 13 years from SCT following heart transplantation and one (#5) died of secondary lung cancer at 20 months from SCT. The early TRM was 24% (95% CI: 4–44%), while the overall nonrelapse mortality was 32% (95% CI: 8–56%).
Nine patients are alive at a median time of 29 months (range 19–116 months). Of these, seven are in hematological and molecular remission at a median time of 50 months (range 19–116 months), whereas two patients (#1, 16) underwent further relapses after SCT, for which both have been treated with antibody-targeted chemotherapy based on mylotarg.
The actuarial probabilities at 10 years of overall survival, disease-free survival and relapse in the whole group of 17 patients were 53% (95% CI: 26–80%), 46% (95% CI: 22–70%) and 33% (95% CI: 6–60%), respectively.
The outcome in terms of RT-PCR monitoring after SCT in the six patients PCR +ve at SCT is illustrated in Figure 1. At the 30–90 day evaluation, two of the six patients had persistence of PCR positivity while four converted to PCR −ve in their BM. Both patients who had tested positive at 30–90 days converted to PCR −ve 1 month after CSA withdrawal. One of these two patients died in molecular remission (#5) and one is alive actually in relapse (#1). Of the other four patients who rapidly converted to PCR −ve after SCT, three (#3, 4 and 6) are in hematologic and molecular remission at 28–80 months, whereas one (#2) has remained PCR −ve for 12 months.
The excellent outcome obtained with ATRA and chemotherapy has questioned the role of allogeneic SCT in APL. However, the procedure is still widely adopted to consolidate patients in 2nd HCR or with more advanced disease. Evaluation of the therapeutic results with SCT and assessment of its efficacy in HCR2 are difficult in light of the few numbers, and large surveys from international registries are needed to obtain significant data.13,14 In this respect, a recent study of the EBMT19 showed significantly lower relapse rate in APL patients transplanted in HCR2 as compared to those receiving autologous SCT, although this benefit was counteracted by higher TRM in the allo-SCT group, resulting in similar overall survival. Interestingly, the relapse risk in the allo-SCT group for patients in HCR2 was similar to that of patients transplanted in HCR1.
One major pitfall in these surveys is that they do not include molecular evaluation of MRD immediately pre-SCT. In these circumstances it is difficult to ascertain whether, prior to transplant, the reported patients were likely destined to further relapse or, alternatively, whether they still had high probabilities of long-term survival. This notwithstanding, the available data clearly suggest that SCT is a valid option for HCR2 APL, while they leave open the question of which type of transplant is preferable for patients who are in molecular remission after salvage therapy.
Although based on a small series, our present analysis included patients prospectively monitored for MRD by a standardized PCR approach carried out in pre-SCT marrow samples as well as post-SCT in order to investigate molecular outcome. This in turn provided a tool for better assessing the effect of SCT, particularly in cases undergoing SCT with molecularly persistent disease. At time of SCT, patients in this study were characterized by advanced disease and poor prognosis. In fact, 16/17 had relapsed after modern, state-of-the-art ATRA plus chemotherapy regimens, 16/17 had received at least two lines of therapy including in some cases autologous SCT and arsenic trioxide, and 6/17 underwent SCT with molecular evidence of disease. The latter group might be regarded as the one associated with poorest prognosis.
In the present series, the overall nonrelapse mortality was considerably influenced by two events (deaths due to second lung cancer and heart transplantation) not directly related to transplant procedure. This finding counterbalanced in terms of survival benefits the efficacy of the allografting, which on the other hand resulted in remarkably low cumulative incidence of relapse. The observation of PCR −ve conversion in 4/6 patients and prolonged molecular remission in all of them further support our interpretation of a strong antileukemic effect of SCT. Finally, both patients who converted to PCR −ve after CSA withdrawal had tested positive at the early evaluation post-SCT, suggesting that a GVL effect, rather than the conditioning regimen, played a major role in this response.
In conclusion, our findings indicate that, in spite of its toxicity, allogeneic SCT is a valid treatment option for APL patients with advanced disease and poor prognosis, being highly effective even in cases with molecularly persistent leukemia, and suggest that a potent GVL effect may take place in APL. Assuming that this is the case, it is hoped that improvements in use of less intensive/immunosuppressive conditioning aimed at reducing TRM while favoring greater GVL effect, might further improve outcome results and survival in APL patients with advanced disease.
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This work was supported by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro), AIL (Associazione Italiana contro le Leucemie), MIUR (Ministero della Università e della Ricerca Scientifica e Tecnologica), CNR (Consiglio Nazionale delle Ricerche) and Ministero della Salute.
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