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Keywords:

  • acute myeloid leukemia;
  • cytogenetics;
  • multicolor flow cytometry;
  • fluorescence in situ hybridization

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

BACKGROUND:

Cytogenetics and multicolor flow cytometry (MFC) are useful tools for monitoring outcome of treatment in acute myeloid leukemia (AML). However, no data are available regarding the meaning of results when the 2 tests do not agree.

METHODS:

The authors of this report analyzed 1464 pairs of concurrent cytogenetics and flow results from 424 patients, before and after hematopoietic cell transplantation, and compared the prognostic impact of discordant and concordant results.

RESULTS:

Informative discordant results were observed in 22% of patients. Compared with patients who had double-negative test results, either positive result had a significant impact on overall survival and relapse-free survival. The hazard ratios with either positive cytogenetic results or positive MFC results pretransplantation were 3.1 (P = .009) and 2.5 (P = .0008), respectively, for reduced overall survival and 2.7 (P = .01) and 4.1 (P < .0001), respectively, for decreased recurrence-free survival. Similar findings were obtained post-transplantation. Molecular cytogenetics, ie, fluorescence in situ hybridization (FISH), added value to the evaluation of discordant cases.

CONCLUSIONS:

The detection of residual AML by either cytogenetics or flow cytometry in patients who underwent hematopoietic cell transplantation predicted early relapse and shortened survival. Cancer 2012;. © 2011 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Cytogenetics and immunophenotyping by flow cytometry are part of the standard evaluation of newly diagnosed acute myeloid leukemia (AML), and both techniques are also used to monitor results of therapy.1-4 Most of the available information about the utility of these techniques to monitor therapeutic response comes from their application after conventional chemotherapy. In that setting, for patients in morphologic complete remission, abnormal cytogenetics predict significantly shorter disease-free survival (DFS) and overall survival.5 However, because of limited sensitivity, the estimated false-negative rate for conventional cytogenetics as an indicator of minimal residual disease (MRD) is approximately 50%; in addition, almost 50% of all patients with AML are cytogenetically normal at diagnosis.3, 6 Molecular cytogenetics with interphase fluorescence in situ hybridization (FISH) has greater sensitivity for the detection of specific chromosome abnormalities, especially for patients with poor chromosome morphology or low or no yield of metaphase cells.7 Exactly how much FISH adds to the predictive power of cytogenetics in monitoring patients with AML after therapy is uncertain.

Flow cytometric methods for MRD detection rely on the degree of immunophenotypic deviation of abnormal populations from normal hematopoietic maturation and, hence, have a variable sensitivity that, for myeloid neoplasms, typically ranges from 0.1% to 0.001% of leukocytes,8 depending on the specific reagent panel used. The advent of high-level multicolor flow cytometry analysis (MFC) allows for the routine detection of up to 10 simultaneous fluorochromes and offers theoretical improvements in both specificity and sensitivity.9 Several studies have demonstrated that the level of MRD is a valuable tool for monitoring continued disease response, detecting early relapse, and predicting patient outcome primarily in the nontransplantation setting.10-12 It should be noted that the lack of standardization for these assays prevents generalizations about assay sensitivity; and, in clinical practice, it is important to inquire about the performance characteristics of the specific assay to be used for MRD assessment.

Because conventional cytogenetics, FISH, and newer, very sensitive MFC techniques are useful for monitoring response to conventional chemotherapy, they are performed increasingly in patients with AML undergoing hematopoietic cell transplantation (HCT). In this setting, we have observed that results from the 3 tests do not always agree. Currently, no data are available to help the physician interpret such discordant results. Although, intuitively, any positive finding should predict for a poor outcome, the sensitivities of current MFC techniques are approaching those of polymerase chain reaction (PCR) analysis; and, at least in the setting of PCR testing for the t(8;21) translocation and for the breakpoint cluster region/c-abl oncogene 1 (BCR/ABL1) fusion transcript in the early post-transplantation setting, positive results have not necessarily been predictive of subsequent relapse.13-15 Because a direct comparison of cytogenetic and MFC results for their prognostic impact in the transplantation setting is not available, especially when the analyses yield discordant results, we conducted a comparison of 1464 paired results from 424 patients with AML who underwent allogeneic HCT. In this study, a positive result from either cytogenetic or MFC testing was associated with a poor prognosis of similar degree. FISH analysis provided further prognostic information in both concordant and discordant cases. These data demonstrated that MRD either pretransplantation or post-transplantation, regardless of how it is measured, predicts outcome.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Patients

Among consecutive patients with AML who underwent allogeneic HCT at the Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance (SCCA) between 2006 and 2009, we evaluated all who had both cytogenetic and flow cytometric results available from the same sample, regardless of specific regimen or treatment protocol. Each patient could have multiple samples tested both pretransplantation and post-transplantation. The results were analyzed for concordance and discordance between the 2 diagnostic methods based on the presence or absence of abnormal cells. In total, 404 patients contributed to the outcome analysis. The study was approved by the Institutional Review Office at the Fred Hutchinson Cancer Research Center/SCCA.

Conventional Cytogenetic Studies

At the time of diagnosis or at pretransplantation and post-transplantation evaluations, samples from bone marrow aspirates were tested for cytogenetic abnormalities using standard culturing and G-banding analysis at SCCA. Karyotype designation was based on the International System for Human Cytogenetic Nomenclature.16, 17 Only clonal abnormalities were considered positive results. The karyotype analysis was based on 20 metaphases for each sample as a routine procedure. The presence of 1 or more clonal abnormalities was considered a positive cytogenetic result; the absence of any clonal aberrations was considered a negative result.

Fluorescence in Situ Hybridization Studies

FISH was performed at SCCA according to standard procedures at the time of testing in a subset of 215 patients. FISH probes were purchased from Abbott-Vysis (Abbott Park, Ill) and Cytocell-Rainbow Scientific (Windsor, Conn). A false-positive cutoff was established for each probe based on the number of abnormal signal patterns observed in 500 cells per control specimen for a total of 20 controls (mean ± 3 standard deviations). An enumeration above the false-positive cutoff was considered a positive result, and an enumeration below the cutoff was considered a negative result.

Flow Cytometry Studies

Ten-color MFC was performed as previously described.18, 19 The panel consisted of 3 tubes as follows: 1) human leukemic antigen-D related (HLA-DR)-Pacific Blue (PB), cluster of differentiation 15 (CD15)-fluorescein isothiocyanate (FITC), CD33-phycoerythrin (PE), CD19-PE-Texas Red (PE-TR), CD117-PE-cyanine 5 (Cy5), CD13-PE-Cy7, CD38-Alexa 594 (A594), CD34-allophycocyanin (APC), CD71-APC-A700, and CD45-APC-H7; 2) HLA-DR-PB, CD64-FITC, CD123-PE, CD4-PE-TR, CD14-PE-Cy5.5, CD13-PE-Cy7, CD38-A594, CD34-APC, CD16-APC-A700, and CD45-APC-H7; and 3) CD56-Alexa 488, CD7-PE, CD5-PE-Cy5, CD33-PE-Cy7, CD38-A594, CD34-APC, and CD45-APC-H7. All antibodies were obtained from Beckman-Coulter (Fullerton, Calif) or Becton-Dickinson (San Jose, Calif). Up to 1 million events per tube were acquired on a custom-built LSRII analyzer, and data compensation and analysis were performed using software developed in our institution. Disease was identified as a population of ≥10 events that deviated from the normal patterns of antigen expression observed in specific cell lineages at specific stages of maturation compared with either normal or regenerating bone marrow. When identified, the abnormal population was quantified as a percentage of the total CD45-positive white cell events. Any level of detectable disease was considered positive.

Statistical Analysis

Overall survival and relapse-free survival were estimated by using the Kaplan-Meier method. For pretransplantation test results, survival was calculated relative to the day of transplantation. For post-transplantation test results, survival was calculated relative to the day of the test. If a patient had multiple test results within a given time window, then the analysis was based on either the first or the last test result as specified below (see Results). Hazard ratio (HR) analyses of survival between groups were performed using Cox regression. For the analysis of recurrence-free survival, patients who relapsed before testing were excluded. Relapse was defined according to standard criteria, such as ≥5% blasts in the bone marrow by morphologic assessment, reappearance of leukemic blasts in the peripheral blood, or reappearance or development of cytologically proven extramedullary disease.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Incidence of Discordant Cytogenetic and Flow Cytometry Results

First, we determined how frequently discordant results were observed. Of 1464 test entries from 424 patients (for patient characteristics, see Table 1), 267 samples (18%) had apparently discordant results, 66% of which were cytogenetically negative (C−) and MFC positive (F+) and 33% were cytogenetically positive and MFC negative (C+/F−). Once patients with normal cytogenetics at diagnosis, loss of chromosome Y only, or known constitutional abnormalities were excluded as noninformative, the incidence of discordant results was 186 samples (12.7%), which accounted for 95 total patients (22%). Among these, 127 samples from 65 patients were C−/F+, whereas 59 samples from 38 patients were C+/F−.

Table 1. Patient Characteristics (n = 424)
CharacteristicNo. of Patients (%)
Median age [range], y50 [0-74]
 0-1733 (8)
 18-60299 (71)
 61-8090 (21)
Sex 
 Male224 (53)
 Female200 (47)
Conditioning 
 Myeloablative288 (68)
 Nonmyeloablative136 (32)
Stem cell source 
 Bone marrow64 (15)
 Peripheral blood311 (73)
 Cord blood49 (12)
Cytogenetic risk pretreatment 
 Good29 (7)
 Intermediate250 (59)
 Poor85 (34)
Status at treatment 
 Remission349 (82)
 Relapse75 (18)

Because MFC analysis can detect abnormalities in as few as 0.001% abnormal cells, whereas cytogenetic analysis has a sensitivity of 5% to 10% for G-banding analysis, the discordant rate was expected to correlate inversely with the frequency of abnormal cells by MFC. Indeed, negative cytogenetic results were identified in 127 MFC-positive samples from 65 patients and had a higher discordant rate when the percentage of abnormal cells detected by MFC was low (Table 2).

Table 2. Discordant Rate Among Patients With Positive Flow Cytometric Results and Negative Cytogenetic Results Based on the Percentage of Abnormal Cells Identified by Flow Cytometric Analysis
  Normal Cytogenetics (Known Abnormal at Diagnosis)
Abnormal Rate by Flow Cytometric Analysis, %No. of SamplesNo.Proportion, %
≥207600
5-<209244.3
1-<5881820
0.1-<1934346
0.01-<0.1684668
0.001-<0.01191684

Negative MFC results in cytogenetically abnormal samples (C+/F−) were identified in 34 patients with aberrations of host origin and in 4 patients with donor-derived abnormalities (mostly deletion of 20q). The majority were low-level positive by cytogenetics; ie, <5 abnormal metaphase cells of 20 total analyzed.

Prognostic Impact of Discordant Results Among Patients With Acute Myeloid Leukemia Who Underwent Transplantation

A primary goal of this study was to determine the impact of discordant results on the overall survival of patients. For the initial analysis, C+ cases included those that were abnormal by G-banding analysis and/or FISH. If a patient had multiple test results within a given time window, then the analysis was based on the last test result for the pretransplantation window and the first test result post-transplantation.

For both pretransplantation and post-transplantation testing, as expected, positive results for both cytogenetics and flow cytometry (C+/F+) predicted a significantly worse outcome than negative results (C−/F−) (Fig. 1; see Table 3). Patients with discordant results (C+/F− or C−/F+) had significantly worse outcomes than patients with negative results (C−/F−). Overall, the prognosis was almost as poor for the patients with discordant results as for the C+/F+ group.

thumbnail image

Figure 1. These Kaplan-Meier plots illustrate (Left) overall survival and (Right) relapse-free survival according to test results obtained (Top) pretransplantation and (Bottom) post-transplantation. The analysis was based on the last test result for the pretransplantation window (days −60 to −1) and the first test result for the post-transplantation window (Days 1 to 100). C+/F− indicates the patients who had abnormal cytogenetic (C) results (including fluorescence in situ hybridization results) but normal flow cytometry (F) results. C−/F+ indicates the patients who had normal cytogenetic results but abnormal flow cytometry results. C+/F+ indicates those who had both abnormal cytogenetic and flow cytometry results. C−/F− indicates those who had normal cytogenetic results and normal flow cytometry results.

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Table 3. Statistical Analysis of Overall Survival and Relapse-Free Survival as a Function of Cytogenetic and Flow Cytometric Testing
 Overall MortalityRelapse and NRMa
Test ResultsHR [95% CI]PHR [95% CI]P
  • Abbreviations: −, negative; +, positive; C, cytogenetic results; CI, confidence interval; F, flow cytometric results; HR hazard ratio; NRM, nonrelapse mortality.

  • a

    For post-transplantation analyses, only relapses subsequent to test results were counted.

Testing before and after transplantation    
 Pretransplant (last value d −60 to d −1)    
  C+/F−, n = 123.1 [1.3-7.2].0092.7 [1.3-6.0].01
  C−/F+, n = 302.5 [1.5-4.4].00084.1 [2.6-6.5]<.0001
  C+/F+, n = 53)3.7 [2.5-5.6]<.00014.2 [2.8-6.2]<.0001
  C−/F−, n = 1911.0 1.0 
 Post-transplant (first value d 1 to d 100)    
  C+/F−, n = 133.1 [1.5-6.5].0022.8 [1.3-5.8].006
  C−/F+, n = 223.2 [1.8-5.5]<.00014.2 [2.5-7.0]<.0001
  C+/F+, n = 267.2 [4.5-12.0]<.00018.1 [5.0-13]<.0001
  C−/F−, n = 2291.0 1.0 
Post-transplantation time frame    
 D 28 (first result d 14 to d 42)    
  C+/F−, n = 123.5 [1.6-7.3].0013.2 [1.5-6.6].002
  C−/F+, n = 193.7 [2.0-6.6]<.00015.0 [2.9-8.7]<.0001
  C+/F+, n = 175.7 [3.2-10.0]<.00015.8 [3.3-10]<.0001
  C−/F−, n = 1761.0 1.0 
 D 80 (first result d 70 to d 100)    
  C+/F−, n = 74.6 [1.8-12.0].0026.4 [2.7-15.0]<.0001
  C−/F+, n = 213.6 [1.9-6.9].00013.7 [2.0-6.0]<.0001
  C+/F+, n = 2511.2 [6.5-19.0]<.000125.6 [13.0-49.0]<.0001
  C−/F−, n = 1771.0 1.0 
 After d 100 (first result d 101 to d 365)    
  C+/F−, n = 613.0 [3.3-50.0].000216.8 [3.9-71.0].0001
  C−/F+, n = 119.4 [3.5-25.0]<.000110.5 [2.6-41.0].0008
  C+/F+, n = 1822.2 [8.9-55.0]<.000141.4 [11-150.0]<.0001
  C−/F−, n = 811.0 1.0 
 After 1 y (first result 1 y to 5 y)    
  C+/F− and C−/F−, n = 62.3 [0.5-10.0].271.5 [0.2-12.0].69
  C+F+, n = 85.7 [1.8-18.0].00378.6 [17-364.0]<.0001
  C−/F−, n = 1071.0 1.0 
Conditioning regimen before and after transplantation    
 Myeloablative pretransplant (last value d −60 to d −1)    
  C+/F−, n = 95.2 [2.0-14.0].00093.8 [1.5-9.9].006
  C/−F+, n = 202.7 [1.3-5.8].015.4 [2.9-9.9]<.0001
  C+/F+, n = 445.6 [3.3-9.4]<.00016.1 [3.7-10.0]<.0001
  C−/F−, n = 1241.0 1.0 
 Myeloablative post-transplant (last value d 1 to d 100)    
  C+/F−, n = 53.8 [0.9-16.0].075.8 [1.8-19.0].004
  C−/F+, n = 142.6 [1.1-6.3].033.0 [1.4-6.5].004
  C+/F+, n = 329.2 [5.4-16.0]<.000116.6 [8.9-31.0]<.0001
  C−/F−, n = 1471.0 1.0 
 Nonmyeloablative pretransplant (last value d −60 to d −1)    
  C+/F−, n = 31.0 [0.1-7.1].971.7 [0.4-7.2].46
  C−/F+, n = 102.5 [1.1-5.4].022.9 [1.4-6.0].005
  C+/F+, n = 92.2 [0.9-5.2].092.4 [1.0-5.8].05
  C−/F−, (n = 671.0 1.0 
 Nonmyeloablative post-transplant (last value d 1 to d 100)    
  C+/F−, n = 62.5 [0.9-7.3].13.5 [1.3-9.4].01
  C−/F+, n = 92.8 [1.2-6.6].024.0 [1.7-9.4].002
  C+/F+, n = 148.1 [3.9-17.0]<.000116.1 [7.2-36.0]<.0001
  C−/F−, n = 631.0 1.0 

The next question we addressed was whether any specific post-transplantation time point carried more weight than the others for prognosis when abnormal testing result(s) were obtained. We divided the post-transplantation testing results into 4 time frames. Typically, patients were scheduled to be evaluated at 28 days, 80 days, and 1 year post-transplantation at our institution. We took the first result from each of the following 4 time frames for analysis: day 28 (first result day 14 to day 42), day 80 (first result day 70 to day 100), after day 100 (first result day 101 to day 365), and after 1 year (first result 1 year to 5 years). In general, each time frame had prognostic impact similar to that of the aggregated post-transplantation results. Detailed statistical results are summarized in Table 3.

Because the benefit of allogeneic HCT is attributable to both the conditioning regimen and a potent graft-versus-leukemia (GVL) effect,20 one question was whether the presence of MRD would have the same prognostic significance in nonmyeloablative (NMA) patients as in myeloablative (MA) patients. We compared the 2 groups in the pretransplantation and post-transplantation settings. The results did not differ significantly between the NMA group and the MA group (Table 3).

The entire analysis also was performed on the subset of patients in remission; ie, with <5% blasts. The results remained the same, although P values were slightly different because of the smaller sample size (data not shown).

Fluorescence in Situ Hybridization Results Add Value to Prognostication of Discordant and Concordant, Negative Cases

FISH studies enhance the detection sensitivity of specific chromosome abnormalities and frequently are used in pretransplantation and post-transplantation patient assessment as an adjunct to cytogenetic testing when a “FISHable” marker is available. Of the 1464 total samples in the current study, 564 samples (38.5%) had concurrent FISH studies performed in a total of 215 patients. We used this subset to address the question of whether FISH results add prognostic value to the samples with either concordant or discordant cytogenetic and MFC results.

Five categories were compared in both the pretransplantation and post-transplantation settings: 1) discordant samples (either C+/F− or C−/F+) with negative FISH results, 2) discordant samples with positive FISH results, 3) concordant negative samples (CF−) with positive FISH results, 4) concordant positive samples (CF+ regardless of FISH results, as positive control), and 5) concordant negative samples (CF− and FISH−, as negative control). Groups 2 and 3 were similar and, thus, were combined. Figure 2 and Table 4 demonstrate that, in both the pretransplantation setting and the post-transplantation setting, patients with positive FISH results had a significantly worse outcome than those with concordant negative results (P ≤ .001); during the post-transplantation period, positive FISH results increased the risk among patients who had either discordant or negative CF results (HR: overall survival, 4.0; recurrence-free survival, 4.8).

thumbnail image

Figure 2. The impact of fluorescence in situ hybridization (FISH) testing results on (Left) overall survival and (Right) relapse-free survival are illustrated according to results that were obtained (Top) pretransplantation and (Bottom) post-transplantation. The analysis was based on the last test result for the pretransplantation window (days −60 to −1) and the first test result for the post-transplantation window (Days 1 to 100). CF− indicates the patients who had concordant negative results for both conventional G-banding cytogenetics (C) and flow cytometry (F). CF+ indicates the patients who had concordant positive results for both conventional cytogenetics and flow cytometry. CFDisc indicates all patients who had discordant (Disc) results between conventional cytogenetics and flow cytometry, including either normal cytogenetics but abnormal flow cytometry results or abnormal cytogenetics but normal flow cytometry results. Additional FISH results are shown as abnormal (FISH+) or normal (FISH−).

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Table 4. Statistical Analysis Results for Overall Survival and Relapse-Free Survival as a Function of Fluorescence in Situ Hybridization Results in Addition to Cytogenetic and Flow Cytometric Testing Results
 Overall MortalityRelapse and NRMa
Test ResultsHR [95% CI]PHR [95% CI]P
  • Abbreviations: −, negative; +, positive; C, cytogenetic results; CI, confidence interval; Disc, discordant results; F, flow cytometric results; FISH, fluorescence in situ hybridization; HR hazard ratio; NRM, nonrelapse mortality.

  • a

    For post-transplantation analyses, only relapses subsequent to test results were counted.

Pretransplant (last value d −60 to d −1)    
 CFDisc FISH+, n = 115.1 [2.3-12.0]<.00015.2 [2.5-11.0]<.0001
 CFDisc FISH−, n = 161.8 [0.6-4.8].272.7 [1.2-5.8].01
 CF+, n = 233.6 [1.8-7.2].00023.6 [1.9-6.9]<.0001
 CF−/FISH−, n = 681.0 1.0 
 Disc FISH+ vs FISH−2.9 [1.0-8.7].062.0 [0.8-4.8].14
Post-transplant (first value d 1 to d 100)    
 CF−/Disc FISH+, n = 77.3 [2.2-25.0].0018.4 [2.8-25.0].0001
 CFDisc FISH−, n = 151.8 [0.8-4.0].131.8 [0.9-3.6].12
 CF+, n = 174.4 [2.3-8.4]<.00016.0 [3.2-11.0]<.0001
 CF−/FISH−, n = 991.0 1.0 
 Disc FISH+ vs FISH−4.0 [1.0-16.0].044.8 [1.4-16.0].01

Prognostic Impact of Minimal Residual Disease Detected by Flow Cytometry Analysis

With increasing sensitivity of MFC, an unanswered question is how prognostically significant are low levels of abnormal results in AML by MFC, especially when cytogenetic/FISH results are normal. We compared 2 groups of post-transplantation patients with negative cytogenetic/FISH results using a 0.1% flow abnormal rate as a division point, because many clinical MFC laboratories do not report positive results lower than 0.1%. The testing time frame was out to 1 year post-transplantation, and the first test result was used if there were multiple results. Although the group with abnormal cells <0.1% (n = 20) appeared to have a slightly better outcome than the >0.1% group (n = 23), the difference was not statistically significant (P > .05). There was no obvious difference in the proportion of high-risk cytogenetic patients between groups.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

Although conventional cytogenetics unarguably are important prognostic markers for AML, they are limited by the requirement for the presence of metaphase cells and, hence, the low detection sensitivity. This limitation is clearly demonstrated by the data presented in Table 2. When disease burden is high, both cytogenetics and MFC can detect the disease and, thus, usually produce concordant results. However, as disease burden decreases, the false-negative rate increases with conventional cytogenetic analysis.

Interphase FISH improves detection sensitivity by approximately 10-fold compared with conventional cytogenetics. It has been demonstrated that FISH is more reliable than morphology for defining complete remisison.21 Therefore, it has been proposed as a valid MRD parameter in patients with AML. However, FISH cannot be performed in all patients; it is only possible and informative if there is a known cytogenetic abnormality that is detectable by a specific FISH probe. Frequently, no FISH probe is available for a given cytogenetic aberration.

MFC for MRD also has been correlated strongly with the clinical course.21 Moreover, MFC can be performed in most patients without specific cytogenetic marker(s) and is the most sensitive of the 3 methods described so far. Although PCR allows the detection of 1 abnormal cell from a million normal cells, it requires an informative marker and, thus, currently cannot be performed in a large subset of patients. The sensitivity of up to 0.001% achieved by the new MFC techniques makes it attractive for MRD detection, which is increasingly important in the risk-adapted management of patients with AML. However, it should be noted that the flow cytometric technique requires an informative immunophenotype, which, although present in the large majority of AML, does not yet allow for a uniformly high level of sensitivity in all cases.

We also investigated how often results might be discordant between cytogenetics, FISH, and MFC. Our data indicated an incidence of 22%, although a more accurate rate of discordance would require results from all patients at initial diagnosis, which generally is not feasible for most tertiary care environments where a large percentage of patients are referred for therapy after diagnosis outside the institution, and pretreatment data may be difficult to obtain or may be of inconsistent quality. In the current study, we provide data demonstrating the prognostic impact of cytogenetic, FISH, and MFC testing results in patients with AML who undergo allogeneic HCT, particularly focusing on those patients who have test results that do not agree. Detection of disease both before and after transplantation predicts a poor outcome (Fig. 1). When both cytogenetic and flow cytometry results were positive (C+/F+), survival was significantly inferior compared with the survival of patients who had negative results. Patients with discordant results both before and after transplantation, ie, either cytogenetically abnormal (C+/F−) or MFC abnormal (C−/F+), had outcomes almost as poor as those of the C+/F+ group (Table 3). In fact, the overlapping 95% confidence interval of the HR between discordant results and C+/F+ results indicated the significant prognostic value of any detectable disease.

Whether and when to intervene are typical questions for an oncologist during post-transplantation follow-up when an abnormal test result is obtained. For patients with chronic myeloid leukemia, PCR detection of the BCR/ABL1 transcript at 3 months after allogeneic HCT has no predictive value, and only at later time points is the test useful.15 In the current study of patients with AML who underwent transplantation, however, the timing of detecting an abnormal result did not appear to be a significant factor for outcome (Table 3). Rather, any time an abnormality is detected by either cytogenetics or MFC, there is an increased risk of subsequent relapse and mortality. Furthermore, some believe it may take longer for a patient who undergoes NMA transplantation to fully benefit from the GVL effect; hence, abnormalities detected early after transplantation may not be as informative as those detected later. We attempted to separate the MA patients and NMA patients into 2 analysis groups for comparison using the last values during the pretransplantation and post-transplantation windows (Table 3). Some of the subgroup comparisons did not have enough statistical power to be truly informative, especially for the NMA group, whose sample size was small. It will be interesting to perform follow-up analyses once sufficient numbers of NMA patients are available for study.

FISH has been proven as a reliable MRD parameter in AML in the nontransplantation setting.21 We further confirmed that FISH was useful in the transplantation setting as well (Fig. 2, Table 4). A positive FISH result in a post-transplantation patient, whether cytogenetics and MFC are both negative or discordant, increases the risks of relapse and mortality by 7.3-fold and 8.4-fold, respectively, compared with patients with negative FISH results along with normal cytogenetics and MFC results. Therefore, FISH testing is valuable when there is an informative marker. It is worth noting that FISH probes have various detection sensitivities because of the nature of the probes and the way each clinical laboratory validates its probes, including the number of cells scored for each sample.

The advantages of MFC—high sensitivity and near universal applicability—make it a desirable tool for MRD monitoring. Its prognostic value has been reported in the pretransplantation setting.19 Our data further demonstrated its utility in predicting outcome both before and after transplantation in the context of comparison with cytogenetics. Although many flow cytometry laboratories do not report abnormal populations below 0.1% in AML, we observed that very low levels of involvement (0.001%-0.1%) were associated with a prognosis comparable to that with higher frequencies of abnormal cells (>0.1%). Although this may reflect the biology of the disease, it also may be caused in part by the difficulty in providing consistent estimates of population frequency in bone marrow samples because of a variable degree of hemodilution by peripheral blood that inevitably is present. It is also possible that the abnormal population identified only represents the portion of the neoplasm that exhibits recognizable deviation from normal patterns of antigen expression and, hence, underestimates the total level of involvement when the MRD frequency is low. A corollary is that the ultimate sensitivity of the technique for MRD detection currently varies between patients, depending on their degree of immunophenotypic aberrancy and the composition of the background populations in which detection is attempted, which is believed to be in the range from 0.1% to 0.01% for most patients. Reduced assay sensitivity in a subset of patients is the likely explanation for the small number of discordant cases in which cytogenetics/FISH results are positive and flow cytometry results are negative, and further methodologic improvements are desirable. Despite these limitations, the positive identification of MRD supports potential medical intervention even when a very low level of abnormality is detected by MFC.

In summary, to our knowledge, our study is the first to examine the prognostic significance of concordant and discordant cytogenetic and flow cytometry test results among the AML transplantation population. The evidence is clear that both abnormal cytogenetic results and/or flow cytometric results before and after transplantation are associated with increased risks of relapse and mortality. How to clinically apply these findings is an obvious question. The finding pretransplantation of MRD by cytogenetics, MFC, or FISH predicts for a poor outcome and raises the question of whether survival may be improved by administering additional therapy pretransplantation, by intensifying the preparative regimen, or by the addition of pre-emptive post-transplantation therapy. Similarly, the detection of MRD post-transplantation predicts for subsequent clinical relapse and provides a setting in which to test various post-transplantation therapies. Although our studies do not answer the question of how to intervene, they do provide tools to better select patients in which to test such interventions and to monitor the results of such treatments.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES

The authors are grateful for research funding from the National Institutes of Health, Bethesda, MD grants P30CA15704, P01CA018029 and P01CA078902. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health nor its subsidiary Institutes and Centers.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SOURCES
  8. REFERENCES
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