Conditioning Regimens

Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML

  • A Corrigendum to this article was published on 06 November 2013

Abstract

Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (allo-HCT) can cure patients with AML in CR. However, relapse after RIC allo-HCT may indicate heterogeneity in the stringency of CR. Strict definition of CR requires no evidence of leukemia by both morphologic and flow cytometric criteria. We re-evaluated 85 AML patients receiving RIC allo-HCT in CR to test if a strict definition of CR had direct implications for the outcome. These patients had leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. Eight (9.4%) had persistent leukemia by flow cytometric criteria at allo-HCT. The patients with immunophenotypic persistent leukemia had a significantly increased relapse (hazard ratio (HR): 3.7; 95% confidence interval (CI): 1.3–10.3, P=0.01) and decreased survival (HR: 2.9; 95% CI: 1.3–6.4, P<0.01) versus 77 patients in CR by both morphology and flow cytometry. However, the pre-allo-HCT bone marrow (BM) blast count (that is, 0–4%) was not significantly associated with risks of relapse or survival. These data indicate the presence of leukemic cells, but not the BM blast count affects survival. A strict morphologic and clinical lab flow cytometric definition of CR predicts outcomes after RIC allo-HCT, and therefore is critical to achieve at transplantation.

Introduction

Allogeneic hematopoietic SCT (allo-HCT) remains the most effective treatment for most patients with AML.1 The two main mechanisms by which allo-HCT can cure AML are through the immunologically based GVL effect and leukemic cell cytoreduction by HCT conditioning.2, 3, 4, 5 Reduced-intensity conditioning (RIC) was introduced to allo-HCT more than a decade ago for patients who are older, had significant co-morbid conditions or poorer performance status.6, 7, 8 The anti-neoplastic potency of these RIC HCT regimens relies primarily on the GVL effect rather than ablating all residual leukemic disease.9 It is clear that patients with active leukemia had more relapse and worse OS after allo-HCT regardless of donor type or patient age.10, 11, 12, 13, 14, 15 Following allo-HCT, 40–60% of AML patients in CR1 enjoy long-term OS, whereas <20% of refractory or relapsed AML patients survive.1, 11 Yet even for allo-HCT during CR, relapse remains the most frequent complication of allo-HCT.16 As relapse has been reported more frequently after RIC allo-HCT compared with myeloablative conditioning (MAC) allo-HCT in patients with AML, this suggests heterogeneity in the interpretation of a CR, which may be more important after RIC allo-HCT.17, 18, 19, 20, 21, 22, 23

The CR definition updated in 200324 clearly requires no evidence of leukemia by flow cytometry in addition to the morphologic remission as reported: ‘The presence of a unique phenotype (by flow cytometry) identical to what was found in the pretreatment specimen (for example, CD34, CD7 coexpression) should be viewed as persistence of leukemia’. Given that relapse occurs in only some patients, though all were in a putative CR at the time of allo-HCT, we evaluated our patients receiving RIC allo-HCT in CR to determine whether the CR criteria were strictly applied at the time of allo-HCT and to determine whether patients with immunophenotypic persistent leukemia had a significantly increased risk of relapse.

Subjects and methods

This retrospective cohort study included 85 consecutive adult patients with AML in CR who received a RIC allo-HCT at the University of Minnesota between April 2000 and March 2012 and had their leukemia immunophenotype documented at diagnosis and analyzed at allo-HCT. We recorded patient BM blast counts from the original clinical flow cytometry and BM morphology reports. Patients with de novo (N=72) or secondary AML (N=13) were included. Pre-transplantation comorbidities were determined retrospectively for all patients using the HCT-specific comorbidity index (HCT-CI)25 and were categorized as low-risk (score 0), intermediate-risk (score 1–2) and high-risk (score 3). Most patients (97%) had intermediate or poor prognostic cytogenetics defined by the Southwest Oncology Group (SWOG) classification.26 Leukemia-free state and CR are defined per International Working Group criteria.24 CCyR was evaluated at the time of HCT and was defined as normal karyotype (by metaphase or FISH analysis), with the disappearance of any cytogenetic aberration detected at the time of diagnosis. Although all patients underwent allo-HCT in CR according to their initial pathology reports, all available BM morphology and flow cytometry data were reviewed by two hematopathologists (SY and MAL) for this study. On review of the flow cytometry data, all patients had flow cytometric data corresponding to their pre-allo-HCT BM biopsy. Eight of these 85 patients (9.4%) had persistent leukemia at the time of allo-HCT, even while the BM morphologic blast count was <5% and no morphologic evidence of leukemia (deemed as a CR).

Transplant types included 54 umbilical cord blood (UCB) (63%), 26 sibling donor BM/PBSC (31%) and 5 other donors (1 mismatched related donor, 3 matched unrelated donors and 1 mismatched unrelated donor (6%). The UCB selection criteria were previously published.27, 28 UCB grafts were matched at 4–6 of 6 HLA-A, -B (Ag level) and -DRB1 (allele level) to the recipient, and in patients receiving two UCB units, similarly matched to each other.

Patients received a RIC regimen including CY (50 mg/kg i.v. on day −6), fludarabine (40 mg/m2 i.v. daily from days −6 through −2) and TBI (200 cGy on day −1) or fludarabine (30 mg/m2 i.v. daily from days −6 through −2) and BU (3.2 mg/kg i.v. daily on days −5 and −4). Equine anti-thymocyte globulin 15 mg/kg i.v. every 12 h for six doses was added for a subgroup of patients (n=19) who had received no chemotherapy within 3 months of allo-HCT. Nearly all (94%) patients received GVHD prophylaxis with CYA (from days −3 to +180) and mycophenolate mofetil (from days −3 to +30). Filgrastim was administered to all patients from day +1 until the ANC was >2.5 × 109/L for 2 days. Treatment protocols were approved by the University of Minnesota Institutional Review Board and registered at clinicaltrials.gov and all patients gave written informed consent before HCT.

Pathology and flow cytometry

All BM biopsies and flow cytometry (all performed within 2–3 weeks before allo-HCT conditioning was started) data were reviewed by hematopathologists at the University of Minnesota Medical Center. In brief, diagnostic and subsequent biopsies received their review at the time of initial consultation with the transplant physician. The majority of data collected for this study were based on BM biopsy and aspirate material collected at the transplant center during the pre-transplant workup assessment. BM aspirates were Wright–Giemsa stained as previously described.29 For a majority of cases, the morphologic blast count was determined by performing 500 cell differential counts on the BM direct aspirate smear, concentrate aspirate smear or touch preparation, and reporting the blast percentage as a total number of nucleated cells. In a minority of cases where the aspirate or touch preparation was not representative of marrow cellularity due to fibrosis or a subcortical sample, blast percentage was estimated by immunostaining the trephine core biopsy for CD34 and/or CD117. The immunohistochemistry was also informative in cases with foci of increased blasts.24

Flow cytometry was performed using the clinical laboratory techniques in place over the 12-year period of study. For one patient, the marrow biopsy obtained prior to transplant was performed externally, and concurrent flow cytometry was not performed. The pathology data received was centrally reviewed and was integrated into the consultative report by the reviewing hematopathologist. Flow cytometry was performed by four-color analysis on either a Becton Dickinson FacsCalibur or a Becton Dickinson FacsCANTO II (Becton Dickinson, San Jose, CA, USA) using Abs conjugated to the following four fluorochromes: FITC, PE, PerCP and APC. Abs used included: CD3, CD7, CD10, CD13, CD14, CD15, CD19, CD33, CD34, CD45, CD56, CD117 and HLA-DR, and were obtained from Becton Dickinson. If sufficient cells were present, 100 000 cells were collected for each tube (typical in most cases). Data were analyzed either by FACSDiva (BD Biosciences, San Jose, CA, USA), FCS Express (De Novo Software, Los Angeles, CA, USA) or Kaluza (Beckman Coulter, Brea, CA, USA). Gating strategies to clean up and isolate cell populations were similar, independent of the software used to analyze the data. A CD45 versus side scatter plot was used to subgate different populations, including lymphocytes, monocytes, granulocytes, blasts and hematogones. Boolean gating was used to look at Ag expression of these different cell populations for all Ags described above in the panels.

Using the CD45-positive cells as the denominator, blast percentage was determined by creating a gate around the region where most blast events occurred, based on their decreased CD45 expression and side scatter. Blast percentage was reflected as a total percentage of the CD45-positive leukocytes, and for this study included non-leukemic and leukemic blasts, if present in the sample. Detectable leukemia (abnormal blast or maturing myeloid population) was identified as a cell population showing deviation from the normal or expected patterns of Ag expression seen on different cell types compared with normal or regenerating marrow samples.

Statistical analysis

Comparison of factors by measures of detectable leukemia was evaluated by the Fisher’s exact test for categorical variables and the Wilcoxon rank-sum test for continuous variables. Blast counts measured by morphology or flow cytometry were tested for their impact on clinical outcomes using cutoff points between 0.5 and 2.5% BM blasts. For the univariate comparisons on OS, Kaplan–Meier curves at 5 years post HCT are reported with comparisons between groups using the log-rank test.30 Cumulative incidence estimates are reported for relapse, treating non-relapse mortality as a competing risk, and the converse for the incidence of non-relapse mortality.31

The independent effect of persistent leukemia by flow cytometry was evaluated in multiple variable regression analyses. Factors evaluated for independent risk or potential confounding effects of persistent leukemia on OS, relapse and non-relapse mortality were blast count (using cutoff points of 0.5 and 2.5%), CMV serostatus (patient-/donor-negative versus patient-negative/donor-positive versus patient-positive), patient age (<60 versus 60 years), patient gender, Karnofsky performance score at baseline (<90 versus 90), conditioning using anti-thymocyte globulin, HCT-CI (low-risk versus intermediate-risk versus high-risk), myelodysplastic syndrome prior to AML, WBC at diagnosis (<20 × 109/L versus 20), time from diagnosis to allo-HCT in patients in CR1 (<6 months versus6 months), length of first remission in patients in CR2 <1 year versus remission 1 year, CR number, cytogenetic risk (high- versus intermediate- versus poor-risk), CNS leukemia pre-transplant and cytogenetic CR at allo-HCT (persistent cytogenetic abnormality versus cytogenetic remission). Cox regression was used to assess the independent effect of persistent leukemia on OS.32 Fine and Gray regression was used to assess the independent effect of persistent leukemia on relapse treating non-event deaths as competing risks.33

Results

The median age of the patients was 60 years (range, 24 to 75 years). Seventy-eight percent of the patients (n=70) had 90% Karnofsky performance score. Sixty-six percent of the patients (n=56) were in CR1. Most patients had intermediate- (n=43, 51%) or poor-risk (n=39, 46%) cytogenetics. Secondary AML was present in 15% (n=13) of the study cohort. The median BM blast counts as assessed by morphology and flow cytometry were 0.6% and 1% at the time of allo-HCT, respectively. Among individual patients, there was a weak correlation between blast counts determined by morphology and flow cytometry (R=0.27). All patients were confirmed by morphologic criteria to be in CR at the time of allo-HCT.

When flow cytometric data corresponding to BM samples at diagnosis and at allo-HCT were reanalyzed, eight patients (9.4%) had persistent leukemia at allo-HCT documented by flow cytometry (Tables 1 and 2). In this example, leukemic blasts clearly had coexpression of CD56 and CD34. Clinical features of these 8 patients with detectable disease and 77 patients who were in a stringent CR are given in Table 2. Despite the limitation of the small number of patients with detectable disease, Fisher’s exact test showed that those with persistent leukemia had a trend toward more frequent poor-risk cytogenetics (P=0.09), higher risk HCT-CI (P=0.11) and higher pre-HCT morphology blast counts (P=0.05). Grade II-IV acute GVHD and chronic GVHD seemed similar between these two groups (Table 2). Importantly, no patients receiving related or URD PBSC had positive pre-HCT persistent leukemia, and all patients with persistent leukemia received UCB grafts. Thus patients receiving UCB grafts were included in a subanalysis. These eight patients with persistent leukemia had a significantly increased relapse incidence at 1 year compared with those who were in a stringent CR (63%; 95% confidence interval (CI): 29–97%) versus 26% (95% CI: 16–36%; P=0.05), respectively (Figure 1). In multivariate analysis, persistent leukemia was associated with a significantly increased relapse rate (hazard ratio (HR): 3.7; 95% CI: 1.3–10.3; P=0.01) (Table 3). CMV-seropositive status was also associated with decreased relapse rates (HR: 0.4; 95% CI: 0.2–0.8; P=0.02) (Table 3). Interestingly, neither blast counts by morphology or cytometry showed an association with relapse rates, non-relapse mortality or OS.

Table 1 Summary of immunophenotypic features of 8 patients who had flow cytometric results that were at least suspicious for persistent leukemia at the time of pre-transplant marrow biopsy
Table 2 Comparison of patients with and without persistent leukemia by flow cytometry
Figure 1
figure1

Patients with no persistent leukemia by flow cytometry had reduced risks of relapse.

Table 3 Multivariate regression analysis showed that persistent leukemia and no CMV seropositivity were associated with increased relapse rates.

Patients with persistent leukemia had decreased OS compared with those in CR (38%; 95% CI: 9–67% vs 64%; 95% CI: 52–74%; P<0.01) at 1 year and at 5 years (0 versus 29%; 95% CI: 14–46%; P<0.01), respectively (Figure 2). In multivariate analysis, persistent leukemia was the only factor that remained as an independent predictor of mortality (HR: 2.9; 95% CI: 1.3–6.4; P<0.01) (Table 3).

Figure 2
figure2

Patients with no persistent leukemia by flow cytometry had improved OS.

In the subanalysis, when the effect of persistent leukemia on OS was evaluated only in patients receiving 54 UCB grafts, persistent leukemia was found to be poorly prognostic (P=0.02). Estimated OS at 100 day, 1 year and 2 years were 75% (45–99%), 38% (4–72%) 0%, respectively, in 8 patients with persistent leukemia, respectively. Estimated OS at 100 days, 1 year and 2 years is 85% (75–95%), 62% (48–76%) and 42% (26–58%) in 46 patients in CR, respectively. Thirty-nine of the 46 patients in stringent CR (85%) received double UCBT, whereas all 8 patients (100%) with persistent disease received double UCT, P=0.30. Between these two groups, median cell doses (regarding total nucleated cell count, CD34+ cell count and CD3+ cell count) were not significantly different. HLA mismatch (that is, HLA matched <5/6) was not significantly different between the stringent CR patients (28%) and the persistent-disease patients (38%), P=0.74. KIR ligand mismatch status was similar as well: 26% (11/43) in the 43 UCB evaluable patients in stringent CR compared with 43% (3/7) in the seven UCB evaluable patients with persistent disease, P=0.30 (KIR mismatch status could not be determined in four patients due to the lack of HLA B and/or C loci data).

No specific morphologic or flow cytometric blast count cutoff point was associated with better or worse outcome. Cytogenetic abnormalities were detected at diagnosis and retested at the time of allo-HCT in 64 patients (41%). Of these 64 patients, 55 (35%) had achieved a CCyR prior to allo-HCT, whereas only 3 (6%) had a persistent abnormal cytogenetic clone. Relapse and OS at 5 years were not statistically different between patients who achieved a CCyR (44 and 34%), those who did not achieve CCyR (56 and 30%) and others (patients with normal cytogenetics or those not tested at both time points; 34 and 36%). In the CR1 subgroup (n=66), treatment with or without consolidation therapy (33 and 32 patients, respectively) did not affect the relapse (5-year estimate was 32% for both; data unavailable in one patient).

Discussion

Our study indicates that when a CR definition is strictly applied as outlined in the defining article,24 it strongly predicts the outcome of RIC allo-HCT. Although the number of patients with detectable leukemia was small in our study population, its impact on OS and relapse were very significant. These patients seemed to have other high-risk factors, including more frequent poor-risk cytogenetics and higher risk HCT-CI; however, in multivariate analysis, persistent leukemia was the only independent factor associated with OS. The sensitivity and specificity of flow cytometry to detect a marker are affected by multiple factors.34 In this study we used standard four-color flow cytometry, and it was re-evaluated by two hematopathologists who were unaware of the survival data, and confirmed the patient’s status as with or without detectable leukemia.

Persistent leukemia can also be detected by other methods. Oran et al.35 showed that persistence of cytogenetic aberrations was associated with increased relapse after MAC allo-HCT. In our study the presence of persistent cytogenetic aberrations did not have an impact on RIC allo-HCT. The difference could be attributable to patient demographics as well as the intensity of pre-allo-HCT conditioning. In addition, Engel et al.36 suggested that persistence of some but not all cytogenetic aberrations increased risks for relapse. In patients with leukemia-associated molecular abnormalities, the leukemia burden can be followed by RQ–PCR, as minimal residual disease, and may be useful to predict relapse with37, 38 or without allo-HCT.39 Minimal residual disease detected using up to 10-color multidimensional flow cytometry has been shown to predict relapse in MAC allo-HCT settings in adults or in children.40, 41

Our study did not find that patients with lower blast counts had favorable outcome compared with those with higher blast counts, while still being in morphologic CR. This suggests that the quantity of non-leukemic myeloblasts has no influence on outcome. However, if leukemic blasts is present—even in small numbers—their presence in the BM (tumor burden) is associated with poorer outcome, irrespective of morphologic blast count.14, 15, 42 This is particularly applicable for RIC allo-HCT.11, 21, 43, 44, 45

We observed a reduced risk of relapse in CMV seropositive recipients. Although we did not evaluate CMV reactivation after allo-HCT, CMV reactivation after allo-HCT has been associated with a decreased risk of relapse,46 possibly due to promotion and persistence of educated natural killer cells.47 All the patients with residual disease (RD) received UCB transplantation, which confounded our analysis by graft source. However, when we analyzed the effect of persistent leukemia limited to the UCB transplant recipients, we found that persistent leukemia was still associated with higher risks of relapse and poorer survival.

The definition of CR (and minimal residual disease or residual disease) is a moving target, especially in light of evolving more sensitive and specific techniques to detect novel markers in AML.39, 48, 49 Our recent international survey revealed significant differences in the definition of CR in AML among physicians depending on their specialty and subspecialty (for example, leukemia physician versus BMT physician and adult physician versus pediatric physician etc.50 It is very likely that the definition of CR will be redefined in the near future.48 However, the standardization of these novel diagnostic tools and their clinical implications will take time. Our study shows that current CR definition—depending upon already standardized (routine) morphologic and flow cytometric analysis—is clinically very useful at least before RIC allo-HCT when applied very strictly. In fact, it is apparent from our own institutional experience that some patients were deemed to be in CR by morphologic criteria despite the presence of persistent leukemia by flow cytometry. Our data suggest that the use of stringent clinical laboratory standard flow cytometry and morphologic evaluations can accurately identify a subgroup of AML patients who are more likely to benefit from RIC allo-HCT. For those at a higher risk, MAC HCT might be considered to improve the leukemic control.2, 51, 52 In patients who do not meet strict CR criteria and so are unfit to undergo a MAC allo-HCT, experimental targeted therapies and/or cellular therapies (for example, tyrosine kinase inhibitors in FLT-3-positive patients, hypomethylating agents53 or haploidentical natural killer cell therapy54) might be preferred over further conventional drug consolidation as a bridging therapy to allo-HCT. Consolidation chemotherapies did not improve allo-HCT outcome before either MAC or RIC transplantation.55, 56 Whether CR is achieved or not after induction therapies critically affects allo-HCT outcome.57 Achieving a stringent CR is critical for RIC allo-HCT success.

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Correspondence to C Ustun.

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Ustun, C., Wiseman, A., DeFor, T. et al. Achieving stringent CR is essential before reduced-intensity conditioning allogeneic hematopoietic cell transplantation in AML. Bone Marrow Transplant 48, 1415–1420 (2013). https://doi.org/10.1038/bmt.2013.124

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Keywords

  • allogeneic hematopoietic cell transplantation
  • reduced-intensity conditioning regimen
  • AML
  • relapse
  • minimal residual disease
  • CR

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