Abstract
Stringent complete remission (CR) in acute myeloid leukemia (AML) requires the absence of both morphologic and flow cytometric evidence of disease. We have previously shown that persistent AML detected by flow cytometry (FC+) before reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (alloHCT) was associated with significantly increased relapse, shorter disease-free survival (DFS) and poorer overall survival (OS), independent of morphologic blast count. We evaluated the effect of FC status on outcomes of alloHCT for AML after either myeloablative conditioning (MAC) or RIC regimens. In 203 patients (MAC, n=80 and RIC, n=123) with no morphologic evidence of persistent AML pre-transplant on marrow biopsy. The allografts included 130 umbilical cord blood (UCB) and 73 sibling donors. We performed central review of pre-transplant standard sensitivity flow cytometry to identify detectable FC+. Twenty-five patients were FC+, including 15 (18.7%) receiving MAC and 10 (8.1%) RIC alloHCT. Among RIC patients FC+ was associated with significantly inferior relapse, disease-free survival (DFS), and overall survival (OS) [multiple regression hazard ratio (HR) 3.8, (95% confidence interval (95% CI) 1.7–8.7), p<0.01 for relapse; HR 2.9, (95% CI: 1.4–5.9), p<0.01 for DFS, and HR 3.4 (95%CI: 1.7–7), p<0.01 for OS]. In contrast, FC+ status was not associated with relapse or decreased OS after MAC. These data suggest that MAC, but not RIC, overcomes the negative effect of pretransplant FC+ following sibling or UCB alloHCT. Therefore, a deeper pre-transplant leukemia-free state is preferred for those treated with RIC.
Keywords: myeloablative conditioning, reduced-intensity conditioning, relapse, survival, allogeneic hematopoietic cell transplantation, flow-cytometry, complete remission, AML
Introduction
Allogeneic hematopoietic stem cell transplantation (alloHCT) is now possible for older patients and those with co-morbid conditions using reduced-intensity conditioning (RIC) protocols.[1–3] Previously published studies have shown that overall survival (OS) and disease-free survival (DFS) after RIC alloHCT for patients with standard or low risk of relapse might be similar to that after myeloablative conditioning (MAC), because the reduced risk of relapse with MAC is offset by its increased non-relapse mortality (NRM).[4–7] This has mostly been studied in patients transplanted in complete remission (CR). In contrast, for patients who are not in CR, MAC has been preferred due to RIC's limited success in the setting of persistent disease.[8] The effect of conditioning intensity on the outcome of AML patients in CR and those not in CR has not been clearly determined. Contributing to this lack of clarity is that the most recent definition of CR was described in 2003.[9] Since then, there have been major advances in our understanding of molecular genetic abnormalities in AML and many advances in the technology of flow cytometric detection of minimal residual disease. However, a standard definition of minimal residual or minimal detectable disease (MRD) has not been established.[10]
We recently showed that achieving stringent CR prior to RIC alloHCT was a prerequisite for better outcomes.[11] While many studies advocate that absence of morphologic evidence of leukemia is compatible with a CR, we had previously suggested that stringent CR includes a leukemia-free state as defined by negative morphology and negative standard sensitivity flow cytometry. In this study, we compared AML patients who received alloHCT with either RIC and MAC regimens and retrospectively applied our suggested stringent CR criteria. The primary goal was to evaluate the association of pre-transplant MRD by standard sensitivity flow cytometry (FC+) with outcomes after either MAC or RIC alloHCT. We hypothesized that the more intensive MAC regimens would lead to improved cytoreduction and better overcome the negative impact of pretansplant FC+.
Patients and Methods
The cohort studied included 208 consecutive adult patients with AML in CR at the time of transplant who received either a MAC or RIC alloHCT (UCB or sibling donor) at the University of Minnesota between January 2003 and January 2014. All had bone marrow (BM) biopsies evaluated both at diagnosis and immediately pre-alloHCT. Patients were consented and treated according to protocols approved by the University of Minnesota Institutional Review Board and registered at clinicaltrials.gov. Data on pre-transplantation comorbidities were collected prospectively and confirmed retrospectively for all patients using the HCT-specific comorbidity index (HCT-CI)[12] and were categorized as low-risk (score 0), intermediate-risk (score 1–2) and high-risk (score ≥3). Cytogenetic data (G-banding and/or FISH analyses) at diagnosis were classified according to the Southwest Oncology Group (SWOG).[13] Leukemia-free state and CR were defined according to the International Working Group criteria.[9] All patients were in CR at the time of alloHCT by initial morphologic and criteria, independent of flow cytometry status. Upon re-review of available hematopathologic data for this study, five patients were found to have morphologic (microscopic) evidence of AML and were therefore excluded. The remaining 203 patients comprised the study group and were evaluated for the presence of flow cytometric evidence of leukemia at the time of alloHCT. The MAC and RIC groups were then classified by the presence (FC+) or absence (FC−) of flow cytometric evidence of leukemia. The outcomes of alloHCT were compared between FC+ and FC− patients in each MAC and RIC cohort. Cytogenetic abnormalities were also evaluated as another mean of evidence of leukemia at the time of alloHCT in patients with abnormal cytogenetics at diagnosis.
Pathology and Flow Cytometry
For this study, all bone marrow biopsy and flow cytometry data (performed within 2 to 4 weeks prior to the alloHCT conditioning) were centrally reviewed by at least one hematopathologist (EC, SY, and/or MAL) at the University of Minnesota Medical Center. In addition, all original data were reviewed by members of the University’s Hematopathology Division at initial presentation. In brief, diagnostic and pre-alloHCT biopsies were reviewed at the initial transplant consult and during the pre-transplant workup assessment. Bone marrow aspirates were Wright-Giemsa stained as previously described.[14] For the majority of cases, the morphologic blast count was determined by performing 500-cell differential counts on the bone marrow direct aspirate smear, concentrate aspirate smear, or touch preparation; blasts were reported as a percentage of the total number of nucleated cells. In a minority of cases (e.g., when 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. Immunohistochemistry was also informative in cases with foci of increased blasts.[9]
Flow cytometry was performed using routine protocols in place over the 12-year period of study. For one patient, the marrow biopsy obtained prior to transplant was performed at an outside institution, and concurrent flow cytometry was not performed. Hematopathologic data received from outside institutions was centrally reviewed and integrated into the consultative report by the reviewing hematopathologist. Flow cytometry was performed by 4-color analysis on either a Becton Dickinson FACSCalibur or a Becton Dickinson FACSCanto II (San Jose, CA), using antibodies conjugated to the following 4 fluorochromes: FITC, PE, PerCP, and APC. Antibodies used included CD3, CD7, CD10, CD13, CD14, CD15, CD19, CD33, CD34, CD45, CD56, CD117, and HLA-DR, and were obtained from Becton Dickinson (San Jose, CA). Sufficient cells were available in most cases to collect at least 100,000 cells for each tube. Assuming a minimum of 100 events were required to comprise an atypical population, the maximum possible sensitivity of this assay would be 0.1%. Data were analyzed either by FACSDiva (Becton Dickinson, San Jose, CA), FCS Express (De Novo Software, Glendale, CA), or Kaluza (Beckman Coulter, Brea, CA); gating strategies were similar regardless of software. A CD45 versus side scatter plot was used to gate different populations, including lymphocytes, monocytes, granulocytes, blasts, and hematogones. Boolean gating was used to look at antigen expression of these different cell populations for all antigens in the above 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 reported as a total percentage of the CD45-positive leukocytes, and for this study included non-leukemic and leukemic blasts (if present). Detectable leukemia (abnormal blasts or abnormal maturing myeloid population) was identified as a cell population showing deviation from the normal or expected patterns of antigen expression seen on different cell types compared to normal or regenerating marrow samples.[15]
Transplantation and Supportive Care
Patients received UCB or sibling grafts. The UCB selection criteria have been previously described. [16, 17] UCB grafts were matched at 4–6 of 6 HLA-A, -B (antigen level) and -DRB1 (allele level) to the recipient, and, in patients receiving two UCB units, were similarly matched to each other.
Patients who underwent an RIC regimen generally received cyclophosphamide (50mg/kg IV on day −6), fludarabine (30–40mg/m2 IV daily from days −6 through −2) and total body irradiation (TBI) (200cGy on day −1) or fludarabine (30mg/m2 IV daily from days −6 through −2) and busulfan (3.2 mg/kg IV daily on days −5 and −4). Equine anti-thymocyte globulin (ATG) 15mg/kg IV every 12h for six doses was added for a subgroup of patients (n=23) who had received no chemotherapy within 3 months of alloHCT. MAC most often included cyclophosphamide (60 mg/kg intravenously daily for 2 days) and 1320 cGy TBI given divided into 8 fractions. The remaining group received busulfan (12.8 mg/kg), fludarabine, and melphalan (n=4) or busulphan/cyclophosphamide (n=3). In MAC regimens, each TBI dose and busulfan dose was higher than myeloablative doses indicated in the CIBMTR conditioning intensity defining article(e.g. TBI ≥800 cGy and busulphan> 8mg/kg).[18]
Graft-versus-host disease (GVHD) prophylaxis for UCB and RIC HCT consisted of cyclosporine (CSA) and mycophenolate mofetil (MMF) or sirolimus plus MMF. MMF was discontinued on day+30. In the remainder, CSA (most often with short-term methotrexate (n=27) was used.
Granulocyte-colony stimulating factor (G-CSF) was administered to all patients from day +1 until the absolute neutrophil count was more than 2.5 × 109/L for 2 days. Institutional standard antimicrobial prophylaxis covering fungal, bacterial, and viral agents were administered in all patients.
Statistical Analysis
Comparison of factors by measures of detectable leukemia was evaluated by the Fisher’s Exact test for categorical variables when appropriate and the Wilcoxon rank-sum test for continuous variables. Correlations between detectable leukemia and blast counts as measured by morphology and flow cytometry were evaluated by comparison of distributions of blasts between the two categories. Statistical comparison of the distributions was completed by the Wilcoxon rank-sum test. Unadjusted Kaplan-Meier curves were used to estimate the probability of OS and DFS with a log-rank test for the comparisons.[19] Cumulative incidence estimates were reported for relapse, treating NRM as a competing risk; and the converse for the incidence of NRM [20].
The independent effect of FC+ was evaluated in multiple regression analyses separately for the MAC and RIC cohorts because the two types of conditioning showed different effects on patients with FC+. Other factors considered were patient age (<60 vs. ≥60 years among RIC and <18 vs. 18–35 vs. >35 among MAC), patient sex, Karnofsky performance score (KPS) (<90 vs. ≥90), cytomegalovirus (CMV) serostatus of the recipient (negative vs. positive), conditioning using ATG, HCT-CI (low-, intermediate- or high-risk), myelodysplastic syndrome (MDS) prior to AML, white blood cell (WBC) count at diagnosis (<20×109/L vs. ≥20), time from diagnosis to alloHCT in patients in first remission (CR1) (< 6 months vs. ≥ 6 months), length of CR1 in patients in second remission (CR2) (< 1 year vs. ≥ 1 year), CR number, and cytogenetic risk (favorable, intermediate or unfavorable). Cox regression was used to assess the independent effect of FC+ on OS and DFS.[21] Fine and Gray regression was used to assess the independent effect of FC+ relapse and NRM.[22] All reported p-values were 2-sided. All analyses were performed using SAS 9.3 (SAS Institute, Inc., Cary, NC) and R version 3.0.2.
Results
Of 203 patients, 80 (39%) received MAC while 123 (61%) received RIC. There were differences in the patient and transplant characteristics between RIC and MAC cohorts (Table 1). ATG was used only in 23 RIC patients. The RIC cohort was older, received alloHCT more recently, more often sirolimus as GVHD prophylaxis, had more intermediate risk cytogenetics, higher morphologic blast counts in bone marrow at the time of alloHCT, lower KPS and higher HCT-CI compared to the MAC cohort.
Table 1.
Patient-characteristics in the RIC and MAC cohorts
| Variable | MAC | RIC | P-value | |
|---|---|---|---|---|
| N | 80 | 123 | ||
| Age (Years) | <0.01 | |||
| Median (range) | 26 (0.6–54) | 61 (25–74) | ||
| Age of Patient | < 18 | 30 (38%) | <0.01 | |
| 18–35 | 21 (26%) | 3 (2%) | ||
| 36–64 | 29 (36%) | 75 (61%) | ||
| >64 | 45 (37%) | |||
| Year of Transplant | 2003–2006 | 17 (21%) | 9 (7%) | <0.01 |
| 2007–2010 | 44 (55%) | 56 (46%) | ||
| 2011–2014 | 19 (24%) | 58 (47%) | ||
| Male | 38 (48%) | 69 (56%) | 0.23 | |
| Donor Type | HLA- Matched Siblings | 26 (33%) | 47 (38%) | 0.21 |
| Single UCB | 9 (11%) | 6 (5%) | ||
| Double UCB | 45 (56%) | 70 (57%) | ||
| Conditioning | Cy/TBI | 23 (29%) | <0.01 | |
| Cy/Flu/TBI | 50 (63%) | 113 (92%) | ||
| Other | 7 (9%) | 10 (8%) | ||
| ATG | 0 | 23 (19%) | <0.01 | |
| GvHD Prophylaxis | CSA/MMF | 54 (68%) | 100 (81%) | <0.01 |
| CSA + Mtx | 26 (33%) | 1 (1%) | ||
| Sirolimus/MMF | 17 (14%) | |||
| Other | 5 (4%) | |||
| Disease Stage | CR1 | 52 | 96 | |
| dx to HCT < 6 months | 48 (60%) | 83 (68%) | 0.14 | |
| dx to HCT >= 6 months | 4 (5%) | 13 (11%) | ||
| CR2 | 28 | 27 | ||
| CR1 < 1 year | 14 (18%) | 12 (10%) | ||
| CR1 >= 1 year | 14 (18%) | 15 (12%) | ||
| Cytogenetic Risk Group | Favorable | 8 (10%) | 3 (2%) | <0.01 |
| Intermediate | 32 (40%) | 80 (65%) | ||
| unfavorable | 36 (45%) | 40 (33%) | ||
| unknown | 4 (5%) | |||
| Recipient CMV+ | 48 (60%) | 81 (66%) | 0.40 | |
| KPS | <90 | 6 (8%) | 22 (18%) | 0.04 |
| HCT-CI score | Low (0) | 48 (60%) | 41 (33%) | <0.01 |
| Intermediate (1–2) | 19 (24%) | 34 (28%) | ||
| High (≥3) | 13 (16%) | 48 (39%) | ||
| BM Blast % Flow Cytometry | Median (range), (IQR) | 1.4 (0–6.0), (1.0–2.0) | 1.0 (0.1–4.0), (1.0–2.0) | 0.14 |
| BM Blast % Morphology | Median (range), (IQR) | 0.9 (0–3.2), (0.4–1.0) | 0.6 (0–4.0), (0.2–1.0) | 0.02 |
| Follow-Up (years) | Median (range), | 5.0 (1.3–9.5) | 3.0 (0.5–7.9) |
Abbreviations: ATG, Antithymocyte globin; CMV, cytomegalovirus; CR, complete remission; CSA, cyclosporine; Cy, cyclophosphamide; dx to HCT, diagnosis to transplant; Flu, fludarabine; GVHD, graft-versus-host disease; HCT-CI, Hematopoietic cell transplantation-specific comorbidity index; KPS, Karnofsky performance status; MMF, mycophenolate mofetil; TBI total body irradiation; UCB, umbilical cord blood
In the MAC and RIC cohorts, 15 (18.7%) and 10 (8.1%) patients, respectively, were FC+ (Table 2). FC+ and FC− patients were similar regarding patient gender, year of alloHCT, GVHD prophylaxis, disease stage, cytogenetic risk group, CMV serostatus, KPS and HCT-CI among all patients, whether receiving MAC or RIC (Table 2). FC+ patients had a higher BM blast percentage by cytometry and morphology compared to FC− patients among all patients, though this trend was statistically significant among MAC, but not RIC patients (Table 2).
Table 2.
Patient-characteristics by FC+ status in the RIC cohort, the MAC cohort, and in all patients
| MAC | RIC | All Patients | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Variable | FC− | FC+ | P-value | FC− | FC+ | P-value | FC− | FC+ | P-value | |
| Number | 65 | 15 | 113 | 10 | 178 | 25 | ||||
| Age (Years) | Median (range) | 25 (0.6–54) | 29 (1–51) | 0.60 | 61 (28–74) | 58 (25–68) | 0.22 | 55 (0.6–74) | 45 (1–68) | 0.01 |
| Year of Transplant | 2003–2006 | 13 (20%) | 4 (27%) | 0.83 | 9 (8%) | 0.23 | 22 (12%) | 4 (16%) | 0.31 | |
| 2007–2010 | 36 (55%) | 8 (53%) | 49 (43%) | 7 (70%) | 85 (48%) | 15 (60%) | ||||
| 2011–2014 | 16 (25%) | 3 (20%) | 55 (49%) | 3 (30%) | 71 (40%) | 6 (24%) | ||||
| Gender: Male | 30 (46%) | 8 (53%) | 0.62 | 63 (56%) | 6 (60%) | 0.80 | 93 (52%) | 14 (56%) | 0.72 | |
| Donor Type | HLA-matched siblings | 22 (34%) | 4 (27%) | 0.48 | 45 (40%) | 2 (20%) | 0.29 | 67 (38%) | 6 (24%) | 0.33 |
| Single UCB | 6 (9%) | 3 (20%) | 6 (5%) | 12 (7%) | 3 (12%) | |||||
| Double UCB | 37 (57%) | 8 (53%) | 62 (55%) | 8 (80%) | 99 (56%) | 16 (64%) | ||||
| Conditioning | Cy/TBI | 19 (29%) | 4 (27%) | 0.78 | 0.82 | |||||
| Cy/Flu/TBI | 41 (63%) | 9 (60%) | 104 (92%) | 9 (90%) | ||||||
| Other | 5 (8%) | 2 (13%) | 9 (8%) | 1 (10%) | ||||||
| ATG | 21 (19%) | 2 (20%) | 0.91 | |||||||
| GvHD Prophylaxis | CSA/MMF | 43 (66%) | 11 (73%) | 0.59 | 91 (81%) | 9 (90%) | 0.45 | |||
| CSA containing | 22 (34%) | 4 (27%) | 1 (1%) | |||||||
| Sirolimus/MMF | 17 (15%) | |||||||||
| Other | 4 (4%) | 1 (10%) | ||||||||
| Disease Stage | CR1, dx < 6 mos | 40 (62%) | 8 (53%) | 0.73 | 76 (67%) | 7 (70%) | 0.52 | 116 (65%) | 15 (60%) | 0.48 |
| CR1, dx ≥6 mos | 3 (5%) | 1 (7%) | 11 (10%) | 2 (20%) | 14 (8%) | 3 (12%) | ||||
| CR2, rem < 1 year | 10 (15%) | 4 (27%) | 11 (10%) | 1 (10%) | 21 (12%) | 5 (20%) | ||||
| CR2, rem1≥1 year | 12 (19%) | 2 (13%) | 15 (13%) | 27 (15%) | 2 (8%) | |||||
| Cytogenetic Risk | good | 7 (11%) | 1 (7%) | 0.56 | 3 (3%) | 0.43 | 10 (6%) | 1 (4%) | 0.79 | |
| intermediate | 24 (37%) | 8 (53%) | 75 (66%) | 5 (50%) | 99 (56%) | 13 (52%) | ||||
| bad | 30 (46%) | 6 (40%) | 35 (31%) | 5 (50%) | 65 (37%) | 11 (44%) | ||||
| unknown | 4 (6%) | 4 (2%) | ||||||||
| Recipient CMV + | 37 (57%) | 11 (73%) | 0.24 | 72 (64%) | 9 (90%) | 0.09 | 109 (61%) | 20 (80%) | 0.07 | |
| KPS | <90 | 5 (8%) | 1 (7%) | 0.89 | 20 (18%) | 2 (20%) | 0.86 | 25 (14%) | 3 (12%) | 0.78 |
| Comorbidity | Low | 36 (55%) | 12 (80%) | 0.17 | 39 (35%) | 2 (20%) | 0.63 | 75 (42%) | 14 (56%) | 0.35 |
| Intermediate | 18 (28%) | 1 (7%) | 31 (27%) | 3 (30%) | 49 (28%) | 4 (16%) | ||||
| High | 11 (17%) | 2 (13%) | 43 (38%) | 5 (50%) | 54 (30%) | 7 (28%) | ||||
| Blasts - Cytometry | Median (range) | 1 (0–5) | 2.5 (1.0–6) | <0.01 | 1 (0.1–4) | 2 (0.5–4) | 0.17 | 1 (0–5) | 2 (0.5–6) | <0.01 |
| Blasts - Morphology | Median (range) | 0.6 (0–3.2) | 1.6 (0–3) | <0.01 | 0.6 (0–4) | 1.1 (0–3) | 0.06 | 0.6 (0–4) | 1.2 (0–3) | <0.01 |
Outcomes
In the RIC cohort, pre-transplant FC+ was associated with significantly higher relapse rates, lower DFS and OS (Table 3B; Figure 1A, 2A, and 3A). Seventy-five percent of FC+ patients relapsed within one year and none of the FC+ patients survived to 2 years. Recipient CMV seropositivity was associated with lower relapse rates, but it was associated with a competing risk of increased NRM. Higher HCT-CI was associated with increased NRM (Table 3B). ATG use did not have an effect on relapse or NRM in univariate analysis.
Table 3.
Multiple regression analysis on outcomes after MAC AlloHCT (A) and after RIC AlloHCT (B).
| DFS | Relapse | NRM | OS | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Factor | N | RR (95% CI) | P | RR (95% CI) | P | RR (95% CI) | P | RR (95% CI) | P |
| FC+ | |||||||||
| No | 65 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| Yes | 15 | 1.1 (0.5–2.5) | 0.79 | 1.3 (0.4–4.1) | 0.60 | 0.6 (0.2–1.9) | 0.42 | 1.1 (0.5–2.4) | 0.85 |
| Age | |||||||||
| <18 | 30 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| 18–35 | 21 | 1.5 (0.7–3.5) | 0.31 | 0.3 (0.1–1.6) | 0.17 | 2.4 (0.9–6.9) | 0.10 | 1.5 (0.7–3.5) | 0.31 |
| >35 | 29 | 2.0 (0.9–4.4) | 0.08 | 1.0 (0.4–2.9) | 0.98 | 2.2 (0.7–6.2) | 0.19 | 2.0 (0.9–4.5) | 0.09 |
| Donor Type | |||||||||
| Matched Sibling | 26 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| UCB | 54 | 1.8 (0.8–3.7) | 0.07 | 0.4 (0.2–1.1) | 0.09 | 4.4 (1.3–14.1) | 0.01 | 2.0 (0.9–4.2) | 0.08 |
| DFS | Relapse | NRM | OS | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Factor | N | RR (95% CI) | P | RR (95% CI) | P | RR (95% CI) | P | RR (95% CI) | P |
| FC+ | |||||||||
| No | 113 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| Yes | 10 | 2.9 (1.4–5.9) | <0.01 | 3.8 (1.7–8.7) | <0.01 | 0.7 (0.2–2.8) | 0.59 | 3.4 (1.7–7.0) | <0.01 |
| HCT-CI | |||||||||
| Low risk | 41 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| Int. risk | 34 | 1.1 (0.6–2.0) | 0.79 | 0.9 (0.4–1.8) | 0.76 | 1.3 (0.4–4.9) | 0.71 | 1.1 (0.6–2.2) | 0.73 |
| High risk | 48 | 1.3 (0.7–2.2) | 0.37 | 0.7 (0.4–1.8) | 0.25 | 3.2 (1.1–8.9) | 0.03 | 1.7 (0.9–3.0) | 0.08 |
| CMV Serostatus | |||||||||
| Negative | 42 | 1.0 | 1.0 | 1.0 | 1.0 | ||||
| Positive | 81 | 0.9 (0.5–1.4) | 0.57 | 0.4 (0.2–0.8) | <0.01 | 4.2 (1.3–13) | 0.02 | 1.0 (0.6–1.7) | 0.90 |
Figure 1.
A. Relapse by Residual Disease-RIC
B. Relapse by Residual Disease - MAC
Figure 2.
A. DFS by Residual Disease- RIC
B. DFS by Residual Disease - MAC
Figure 3.
A. OS by Residual Disease - RIC
B. OS by Residual Disease - MAC
In the MAC cohort, FC status had no statistically significant association with either relapse, NRM, DFS or OS (Table 3A, Figure 1B, 2B, and 3B). However, UCB vs. sibling donor transplantation was associated with increased NRM (Table 3A).
Cytogenetic Evidence of Leukemia
Of 87 patients with abnormal cytogenetics at diagnosis, 85 also had cytogenetic data at the time of alloHCT: 74 had normal cytogenetic (Cy−) and 11 had still evidence of leukemia (Cy+). In these 85 patients, there was a correlation between FC and cytogenetic status: 67 of 72 FC− patients were also Cy− (i.e., negative predictive value of 93%), in contrast only 7 of 13 (54%) FC+ patients were Cy−, p<0.01. There was no effect of Cy+ on relapse, NRM, DFS or OS in each MAC or RIC cohort. However, this might be resulted from low number of Cy+ patients (n=6 in MAC and n=5 in RIC).
Discussion
In this study we show that even in patients with morphologic CR, any evidence of residual leukemia by standard sensitivity flow cytometry is independently associated with a poor prognosis for those receiving RIC (in fact all patients died, most after relapse), but not MAC alloHCT, suggesting MAC can overcome the negative impact of FC+ detected within a remission marrow. A recent report from Seattle showed that FC+ patients had a poor OS (HR 2.69; 95% CI 1.78–4.07), largely due to increased relapse (HR 4.56; 95% CI 2.84–7.34).[23] However, the negative effect of FC+ was more prominent in patients receiving MAC (76% OS at 3 year in FC+ vs. 25% in FC−) than nonmyeloablative (NMA) conditioning (48% OS at 3 year in FC+ vs. 41% in FC−).[23] Relapse rates at 3 years were increased in both NMA (FC+ 57% vs. FC− 28%) and MAC transplants (FC+ 63% vs. FC− 22%).[23] The implications of an FC+ status before MAC transplantation is controversial, with both worrisome,[24] and less concerning reports.[25, 26] A pediatric study showed in morphologic CR, the negative effect of MRD could be overcome by MAC in AML, but not in acute lymphoblastic leukemia.[27]
Walter et al used NMA, rather than RIC which has been reported to yield greater risks of relapse1. In addition this earlier report included URD, but not UCB transplantation. Differing donor sources (such as UCB) may confer differential protection against relapse, as we have observed following MAC.[28] However, in the current analysis, UCB had no effect on relapse, DFS or OS in either the RIC or MAC cohorts. Moreover, previous studies comparing our UCB transplantation with sibling and URD transplantation also showed that survival after UCB transplantation was comparable to other donor types.[3, 29, 30]
A key difference between the current study and others is the difference in flow cytometric methodology. There is no FDA approved method for residual disease testing by flow cytometry, and the analytic sensitivity of tests varies greatly among labs.[10] The majority of labs that perform AML MRD analysis have a maximum lower limit of detection of 0.1% (herein described as standard sensitivity flow cytometry).[10] The Seattle study used a higher sensitivity 10-color method with a lower limit of detection less than 0.1%.[23] During CR, Walter et al observed an FC+ status in 24% of RIC and 19% of MAC patients[23] versus our finding of 18.7% of RIC and 8.1% of MAC patients. Technical factors may influence the measures of MRD and thus the clinical implications for the anticipated outcome.
Only RIC patients received ATG in our study. However, because the use of ATG was similar between FC+ and FC− patients, this cannot explain the significant difference in relapse rates within the RIC cohort. In addition, the effect of ATG on relapse after alloHCT has been controversial, with more recent studies indicating that ATG use does not increase the risk of relapse.[31–34] In our study, ATG use did not have effect on relapse. Recipient CMV seropositivity has been shown to be associated with decreased relapse in AML.[35–37] In this study, we also showed that CMV+ patients had decreased relapse but increased NRM, and thus had similar DFS and OS in the RIC cohort. However, the effect of FC+ on relapse was independent of CMV seropositivity in the RIC cohort.
We compared patient- and disease characteristics between FC+ and FC− patients. FC+ patients were younger and had a slightly higher percentage of blasts in the BM. We did not find a correlation between FC positivity and any other characteristics, including cytogenetic findings, time to transplantation, or disease stage. In the Walter et al study, FC+ patients were more likely to have AML with unfavorable cytogenetics, and also had a higher prevalence of secondary AML, a shorter time between CR to alloHCT, and received fewer courses of consolidation chemotherapy.[23] However, age was similar between FC+ and FC− patients.[23]
FC status was correlated with Cy status in our study; however, its negative predictive value is much higher (93%) than its positive predictive value. This is consistent with Fang et al study.[38] Although we did not observe the negative effect of Cy+ on outcomes in each RIC or MAC cohort, this might be because of limited number of Cy+ patients in the study. Studies from MD Anderson showed negative impact of persistent cytogenetic abnormalities at alloHCT.[38, 39]
There is a need to standardize the definition of response in AML as well as for standardization of flow cytometry techniques.[10] Our study indicates that a stringent CR definition proposed 12 years ago, which defines a morphologic leukemia-free state as no morphologic or standard sensitivity flow cytometric evidence of leukemia, can successfully predict outcomes of alloHCT, especially those receiving RIC. We suggest that FC+ status leads to a poor prognosis for RIC alloHCT patients. Future steps will be to determine prospectively if anti-leukemic interventions, such as additional chemotherapy to reduce pre-HCT MRD, post HCT consolidation, or maintenance therapies based on FC+ status, will improve overall outcome.
Footnotes
Author Contribution: C.U, D.W., M.L. conceived the study idea; E.C., M.L., S.Y. performed pathologic examinations and analysis; T.D. performed statistical analysis, N.R, M.D., E.W., T.D., C.U. did data collection; C.U, M.L., C.B., E.W., N.B. did literature search; all authors contributed to write the article, edited the last version of the article, and agreed with the current version of the manuscript.
Authors have no conflicts of interest to disclose relevant to the material presented in this study.
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