Estimation of relapse risk in AML after allo-SCT is critical. The negative impact of increased blast count post transplant is widely accepted. Here, we studied cellularity and dysplasia in BM cytomorphology on days 30 and 100 in 112 AML patients who achieved haematological CR after SCT. Overall cellularity on day 30 was normal in 45.3%, reduced in 37.3% and increased in 17.3% of samples (day 100: normal: 54.8%; reduced: 38.7%; and increased: 6.5%). Dysplasia in ⩾10% of cells was frequent on day 30 (granulopoiesis: 25.0% of samples; erythropoiesis: 34.6%; and megakaryopoiesis: 47.7%) and also on day 100. Relapses were less frequent in patients with normal BM cellularity on day 30 (7/34; 20.6%) when compared with reduced (9/28; 32.1%) or increased cellularity (10/13; 76.9%; P=0.001). Estimated 2-year OS was 59.0% for patients with normal overall cellularity, followed by patients with increased (44.0%) and reduced cellularity (31.4%, P=0.009). In contrast, cellularity at day 100 and dysplasia at days 30 and 100 did not correlate with outcome measures. Thus, in the cohort studied, BM cellularity represents a prognostic parameter for the post-transplant period in AML patients. Dysplasia seems to be an unspecific phenomenon in the cohort analysed.
Allo-SCT is a curative option for patients with high-risk AML. Relapse risk after SCT depends on initial disease features, such as the karyotype or the pre-transplant disease status, and ranges between 20 and 60%.1, 2 Monitoring minimal residual disease and donor chimaerism analysis after SCT for AML is used to guide immunosuppression, preemptive immunotherapy and administration of chemotherapy or demethylating agents post transplant.3, 4, 5, 6 For some patients with cytogenetically normal AML, disease-specific minimal residual disease parameters are available for the post-transplant period, for example, NPM1 mutations.7, 8, 9, 10 Measurement of WT111 and chimaerism12 have both been used to direct preemptive therapy with donor lymphocyte infusions post transplant. Analysis of WT1 expression has also been combined with chimaerism analysis to identify patients at an increased relapse risk post transplant.13 Once relapse is cytomorphologically manifest, intervention, for example, by adoptive immunotherapy, is considered to be less effective.2, 3 Therefore, it appears desirable to identify patients at higher risk of relapse at an early stage.
Following any therapeutic intervention against AML, failure to achieve blast clearance <5% BM nuclear cells is associated with increased probability of relapse and reduced survival.14, 15 ‘Dyshaematopoiesis’ after allo-SCT, especially in the megakaryocytic lineage, is a frequent sequel of toxic treatment and must not be regarded neoplastic.16 BM fibrosis after allogeneic SCT for myelodysplastic syndromes and AML with multilineage dysplasia has been associated with inferior prognosis in patients with high-risk disease.17 To deepen insights into the impact of cytomorphological parameters such as dysplasia and cellularity in the post-transplant period, we studied 112 stem cell recipients with AML by cytomorphology on days 30 and 100 and performed correlations with survival outcomes.
Materials and methods
Patients transplanted at Hamburg University between 2005 and 2010 were included into this analysis if they met the following criteria: (i) diagnosis of AML, (ii) blast clearance <5% in the BM14 following allogeneic SCT, (iii) availability of BM cytomorphology from at least one time point post transplant (day 30 or day 100). Patients with persistent AML or manifest relapse (⩾5% myeloblasts) before day 30 were excluded. Patients who developed relapse between day 30 and 100 were only evaluated for day 30.
Conditioning was classified as myeloablative conditioning or reduced intensity conditioning according to the EBMT MEDAB manual (http://www.ebmt.org/4registry/Registry3.html, version 21 February 2011). A CMV IgG-seropositive patient was defined to be at ‘high risk’ for CMV reactivation when the donor was CMV IgG seronegative.
BM aspirates were stained by the May Grünwald Giemsa method. A total of 200 nucleated cells was assessed in each BM sample. We evaluated overall BM cellularity and cellularity separately in granulopoiesis, erythropoiesis and megakaryopoiesis.18 Performing age-related adjustment,19 the patients were assigned to the categories increased, normal and decreased cellularity. Examples of each one hypocellular (Supplementary Figures S1a and b) and one normocellular BM smear (Supplementary Figures S2a and b) are supplied. Pictures were taken on an Axio Imager A1 (Carl Zeiss, Jena, Germany) using a 1080i, 3CCD camera (Sony Corp., Tokyo, Japan) by 10-fold (Supplementary Figures S1a and S2a) and 40-fold (Supplementary Figures S1b and S2b) magnification. Dysplasia was evaluated in the different haematopoietic lineages applying the criteria of Goasguen et al.20 and the WHO criteria.21 Thresholds of 10, 20 and 50% of dysplastic cells were applied to categorize any haematopoietic lineage as ‘dysplastic’. BM specimens were analysed by different investigators (MC, KM, MB and PS) and were reviewed by UB. Investigators and reviewer were blinded for identity and clinical course of the patient whose sample was analyzed.
Risk categorization of cytogenetics was performed according to the revised Medical Research Council criteria. The favourable subgroup consists of t(15;17)(q22;q21), t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22). The intermediate subgroup comprises entities not classified as favourable or adverse, and the adverse subgroup is abn(3q) (excluding t(3;5)(q21 25;q31;q35), inv(3)(q21q26)/t(3;3)(q21;q26)), add(5q), del(5q), −5, −7, add(7q), del(7q), t(6;11)(q27;q23), t(10;11)(p11-13;q23), t(11q23) (excluding t(9;11)(p21 22;q23) and t(11;19)(q23;p13)), t(9;22)(q34;q11), −17, abn(17p), complex (four or more unrelated abnormalities).22
Chimaerism analysis was performed by quantitative real-time PCR for donor/recipient-specific polymorphisms (short insertions/deletions),23 and by Y-chromosome-specific sequences24 for recipients of sex-mismatched allografts. The following categories were used: ‘complete donor chimaerism’ (donor-specific polymorphisms in >99% of the patient’s alleles), ‘mixed chimaerism with high donor-allele proportion’ (donor alleles between 90 and 99%), and ‘mixed chimaerism with low donor-allele proportion’ (donor alleles <90%). Chimaerism analysis was performed in parallel to the first BM control (day 30) and was repeated every other week from peripheral blood.
Categorical variables were compared using the Pearson χ2-test. Continuous variables were compared using the Mann–Whitney test. Tests were performed two-tailed and exact. Probabilities of OS were estimated by the method of Kaplan–Meier.25 Probabilities of non-relapse mortality (NRM) and relapse incidence (RI) when competing with each other were computed using the cumulative incidence method.26 Association of cytomorphological and clinical factors with OS was assessed with the log-rank test. Variables with potential impact on univariate analysis were entered into proportional hazards regression analysis.27 The statistical influence of cytomorphological parameters on NRM and RI was analysed using a subdistributive hazard ratio (HR) model.28 PASW/ SPSS version 18.0 (IBM, New York, USA) was used. The cumulative incidence analysis and subdistributive HR model analysis were performed with R statistics software package.29, 30
The basis for this study were 112 adult patients with AML (60 males, 52 females; median, 53 years; range, 17–72 years) who received a 1st (n=101) or ⩾2nd SCT (n=11). Seventy patients suffered from first manifestation of AML, 30 showed the first and 7 showed the second relapse. Disease stage before transplantation was not available in five cases. Karyotype was available in 107 patients. According to the revised Medical Research Council criteria,22 most patients were in the intermediate (n=65) or adverse cytogenetic risk groups (n=35), and only seven were assigned to the favourable group. Information on the pre-transplant remission status was available in 111 patients, with a total of 55 patients in haematological CR, 44 with active disease, whereas 12 were therapy-naïve and received an up-front transplantation including the ‘FLAMSA’ protocol as part of the conditioning regimen.31, 32
Donor and transplantation characteristics
The majority of patients received PBSC as graft source (n=106; BM: n=4; cord blood: n=1; unknown: n=1). In most cases, donors were 10/10 HLA-identical (n=67; mismatched: n=45) and unrelated (n=87; related: n=25). A total of 75/112 patients were CMV IgG-seropositive, 36/112 were seronegative (unknown: n=1). Conditioning regimens were of reduced intensity in 72 cases, mostly following the FLAMSA regimen consisting of fludarabine, cytarabine and amsacrine according to Schmid et al.31, 32 with BU (⩽10 mg/kg b.w. p.o. or equivalent i.v.) or reduced TBI (⩽8 Gy). Myeloablative conditioning was chosen for 40 patients, either based on BU (14 mg/kg b.w. p.o. or equivalent i.v.) or on TBI (12 Gy). A total of 67 patients received antithymocyte globulin. Prophylaxis of GVHD was mainly CYA-based, most frequently combined with mycophenolic acid (n=61) or MTX ( n=48). Acute GVHD was experienced by 62 patients (grade II–IV: n=40) and chronic GVHD by 37 patients (limited: n=25 and extended: n=12). Full donor chimaerism on day 30 was achieved in 84 out of 98 evaluable (85.7%) patients (⩾90% of donor alleles: n=12 and <90% of donor alleles: n=2) and in 73/90 (81.1%) patients investigated on day 100 (⩾90% of donor alleles: n=9 and <90% of donor alleles: n=8). Detailed characteristics of patients, donors and transplant setting are shown in Table 1.
BM cytomorphology was available from 75/112 patients on day 30 post transplant. On day 100, only 62 patients were considered for calculations, as 13 patients were removed from the analysis due to relapse between first and second BM analysis.
At the first BM control (day 30), aberrant cellularity was frequent with increased cellularity in 13 (17.3%) patients and reduced cellularity in 28 (37.3%), whereas normal cellularity was achieved in only 34/75 patients (45.3%) with available morphology. In more detail, granulopoiesis was increased in 15 patients (20.0%) and reduced in 25 (33.3%) patients. Erythropoietic cellularity was increased in 6 (8.0%) and reduced in 36 (48.0%) patients. Megakaryopoiesis was reduced in the majority of BM samples (n=40/75, 53.3%) and increased in 15 (20.0%). Mean peripheral blood leukocyte count in the group with a hypocellular BM at day 30 was 3.74 Gpt/L (95% confidence interval (CI) for mean 1.31–6.17 Gpt/L), whereas in the group with a normocellular BM it was 7.01 Gpt/L (95% CI for mean 4.33–9.69 Gpt/L, P=0.075).
On day 100, BM cellularity was frequently aberrant (increased cellularity: n=4, 6.5% and reduced: n=24, 38.7%). Aberrant cellularity occurred in all the three haematopoietic lineages (shown in Table 2a in detail). Taken together, after allogeneic SCT, both increased and decreased cellularity of BM aspirates were frequent phenomena.
On day 30, dysplastic features in ⩾10% of cells were most frequently observed in megakaryopoiesis with 21/44 (47.7%) patients evaluable for dysplasia in this haematopoietic lineage affected. This was followed by erythropoiesis with 18/52 (34.6%) patients and granulopoiesis with 15/60 (25.0%) of evaluable cases. On day 100, granulopoiesis was dysplastic (⩾10% of cells) in 18/58 evaluable patients (31.0%), erythropoiesis in 24/55 (43.6%) and megakaryopoiesis in 33/52 (63.5%). Data are summarized in Table 2b. Taken together, dysplasia even to a substantial degree was a common phenomenon in all the three haematopoietic lineages of the patients described in this analysis.
Correlation of cytomorphological factors between day 30 and 100
Overall cellularity on days 30 and 100 showed a significant correlation with each other (P=0.006). All other cytomorphological parameters evaluated showed no significant correlations on days 30 and 100, respectively (data not shown).
Correlation of cytomorphological factors with core clinical factors
Subsequently, we evaluated whether patient-specific factors (age and pre-transplant remission status), disease-specific parameters (cytogenetic risk group), transplant-specific characteristics (CMV-status and -matching, related vs unrelated donor, donor sex, donor/patient sex-match, HLA-match, AB0-match, myeloablative vs reduced intensity conditioning, use of antithymocyte globulin, use of TBI, use of type of immunosuppression), or post-transplant outcomes (occurrence of acute or chronic GvHD, leukocyte recovery, platelet recovery and donor chimaerism) correlated with cellularity or dysplasia on days 30 and 100. However, Pearson’s χ2-test and, where applicable, calculation of risk estimates showed no clear correlations of these parameters with these cytomorphological parameters (Supplementary Table S1).
After a median follow-up of 394 days (range, 37–2118 days), estimated 2-year OS was 45.8%. Relapse occurred in 39/112 patients, and 34 of these patients died of relapse. Non-relapse causes (infectious diseases, organ toxicities and severe GVHD) occurred in 19/112 patients. With RI and NRM calculated as competing risks, cumulative incidences of RI and NRM at 2 years were 34% (95% CI, 24–44%) and 17% (95% CI, 9–25%), respectively (Figure 1).
Survival with regards to cellularity
Relapses were less frequent in patients with normal BM cellularity on day 30 (7/34; 20.6%) when compared with those with reduced (9/28; 32.1%) or increased cellularity (10/13; 76.9%; P=0.001). Reduced overall cellularity on day 30 significantly correlated with inferior 2-year OS as compared with increased or normal cellularity (31.4% vs 44.0% vs 59%, respectively; P=0.009; Figure 2a). Separate analysis of cellularity in granulopoiesis (Figure 2b), erythropoiesis (Figure 2c) or megakaryopoiesis (Figure 2d) showed a correlation of reduced cellularity on day 30 with inferior rates of OS (Table 3).
Competing risk-factor analysis revealed that the group of patients with increased cellularity had a cumulative RI of 62% (95% CI 35–89%) with a HR of 6.68 (95% CI 5.7–7.66, P=0.00014, Figure 3). Patients with reduced cellularity had an NRM of 36% (95% CI 18%–54%) with a HR of 2.6 (95% CI 1.6–3.6, P=0.006, Figure 3).
Cellularity of the BM specimen on day 100 showed no significant correlations with overall survival (data not shown).
Survival with regards to dysplasia
Dysplasia in 10% or more cells in any haematopoietic lineage showed no significant correlations with survival outcomes/relapse rate on days 30 (Table 3) and 100 (data not shown). Similarly, thresholds of 20 or 50% of dysplastic cells did not significantly correlate with survival outcomes/relapse rate.
Other prognostic parameters
In the whole cohort, initial manifestation vs relapse of the AML (P=0.028), the achievement of haematological CR before SCT (P=0.007), absence of acute GVHD grade II–IV (P=0.042) and presence of chronic GVHD (P=0.001) were associated with improved 2-year Kaplan–Meier OS estimates (Supplementary Table S2). Other parameters (for example, patient sex, history of the AML, previous chemotherapy, the cytogenetic risk group according to the MRC criteria,22 donor type, HLA-match, the CMV risk constellation or application of antithymocyte globulin) had no significant impact on survival outcomes. When multivariate analysis was performed including the most significant clinical parameters (that is, the stage of disease, the remission status before SCT, severe acute GVHD and chronic GVHD) and cellularity of the BM sample on day 30 as the cytomorphological parameter with the strongest prognostic impact (Table 3), absence of chronic GVHD (HR 0.296, 95% CI 0.129–0.682, P=0.004) and absence of severe acute GVHD (HR 1.347, 95% CI 1.008–1.801, P=0.044) remained independent prognostic parameters (Supplementary Table S3).
Treatment of relapse of AML after allogeneic SCT results in 2-year OS of <20%, for example, when salvage therapy is a second allogeneic SCT.33 A prognostic value has been attributed to quantitative lymphocyte and neutrophil recovery, especially for myeloid neoplasia after allogeneic SCT.34, 35, 36 Attempts are made to diagnose2, 3 and treat11, 12 incipient relapse based on parameters such as NPM1 mutations,7, 9, 10, 37, 38 WT1 overexpression37, 38, 39, 40 and donor–patient chimaerism41, 42, 43 or combinations of those.13 Yet, apart from chimaerism, those methods are frequently available for a limited set of patients (for example, NPM1 mutations). Furthermore, administration of donor lymphocyte infusions on a preemptive11, 12 or prophylactic31 basis aims at selecting patients at risk with high specificity. This applies for other interventions such as the use of demethylating agents.5, 44, 45, 46 Thus, we attempted to add a readily available feature to contribute to other methods of risk assessment. One large study has addressed histological features in myelodysplastic syndromes and AML patients 28, 56, and 84 days after allogeneic SCT and found an adverse association of fibrosis with survival in patients with high-risk disease.17 Here, we wished to complement the knowledge about the frequency and the specific value of cytomorphological parameters other than blast count after allogeneic SCT for patients with AML.
Clearly, our data show that aberrant cellularity and dysplasia are frequent events in patients with AML in the post-transplant period. Cellularity was reduced in 30–50% of cases and dysplasia was present in up to 64% of cases (megakaryopoiesis on day 100). In addition, we found reduced cellularity to be associated with inferior OS when compared with patients who achieved normal cellularity in the first BM aspiration (day 30) post transplant. This finding held true even when the haematopoietic lineages were investigated separately at this time point (Figure 2). In a competing risk-factor model, increased cellularity had a negative impact on OS due to a statistical association with increased RI, and reduced cellularity did so as a result of its adverse impact on NRM (Figure 3). In contrast, dysplasia, although frequent in the post-transplant period, for example, due to the influence of myelotoxic medication, had no significant impact on survival outcomes in the post-transplant period in this study. This was irrespective of the thresholds of dysplastic cells used for correlations (Table 3). Based on our analysis, we hypothesize that BM cellularity is an additional prognostic indicator for patients with AML after allo-SCT. It remains obscure if variation in cellularity of a BM aspiration specimen is a consequence of the malignancy still present below microscopic levels. Pronounced hypocellularity might also well be an epiphenomenon of different features, for example, leading to delayed engraftment and delayed or suspended GVL effect. The results of correlations performed in this analysis are too weak to allow for any hypothesis (Supplementary Table S1). As is the case for the causal analysis of delayed lymphocyte and neutrophil recovery,34, 35, 36 we cannot comment whether, for example, hypocellularity is a matter of increased destruction or decreased production of haematopoietic stem cells and progenitors. Yet, if the trend of a decreased peripheral blood leukocyte count associated with hypocellular BM smears that we observed held true, these phenomena might well be dependent. Problems of this type of analysis such as inter-observer variability should not be neglected. Standardization of morphological evaluation of BM samples is necessary albeit difficult.18, 19 Patient selection can be criticized for being arbitrary, that is, including only patients that had BM samples on day 30 and/or 100. To further characterise the population studied, correlation of clinical factors with 2-year OS was carried out (Supplementary Tables S2 and S3) and confirmed the prognostic value, for example, of chronic GVHD, the stage of disease, the remission status pre-transplant or the absence of acute GVHD above grade I. Thus, the results within our chosen cohort recapitulate results found elsewhere. Our data suggest including BM cellularity of the BM aspirate on day 30 post transplant into possible workups to identify AML patients at increased risk for relapse or NRM post transplant. The prognostic impact of our findings needs further evaluation, both alone and in combination with other parameters, for example, donor chimaerism and minimal residual disease status. Trephine biopsies might be of additional value, especially in order to delineate reasons for hypocellularity. Immediate clinical implications of the presence of a sample with hypocellularity may include an increased surveillance for relapse, for example, through more frequent measurement of minimal residual disease and chimaerism. The therapeutic usefulness of our findings needs to be prospectively addressed.
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We acknowledge Waltraud Schultz, University Cancer Centre Hamburg, for expert technical assistance (cytomorphology). We also acknowledge Dr Michael Roth MD, Department of Pediatrics, Montefiore Hospital and Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA, for critical comments, thoughtful suggestions on data interpretation and critical reading of the manuscript.
Author contributions: MC and UB designed the study and wrote the manuscript. MC, KM, MB, PS and UB performed cytomorphology. KM, TZ, MB and UB assembled data. MC, EK and UB carried out statistical analysis. FA, CB and NK contributed patients. TH and NK contributed to the interpretation of data. All authors contributed to data analysis, reviewed the manuscript and approved its final version.
Data have been assembled during the course of the doctoral thesis of KM. MC has received honoraria and travel support from Celgene. TH declares part ownership of the Munich Leukemia Laboratory GmbH. All other authors have no conflict of interest to declare.
Supplementary Information accompanies the paper on Bone Marrow Transplantation website
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Christopeit, M., Miersch, K., Klyuchnikov, E. et al. Evaluation of BM cytomorphology after allo-SCT in patients with AML. Bone Marrow Transplant 47, 1538–1544 (2012). https://doi.org/10.1038/bmt.2012.70
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