Name: comprehensive protocol to sample and process bone marrow for measuring measurable residual disease and leukemic stem cells in acute myeloid leukemia
Uploaded: 2018-03-05T12:30:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia at JoVE.c

This research has been supported by Dutch Cancer Society (ALPE 2013-6371) and Egbers foundation for VONK. We thank the Dutch AML-MRD working group for the collaboration and fruitful discussions for continuous improvement and standardization of AML-MRD.

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+disease+detected+by+multidimensional+flow+cytometry+signifies+high+relapse+risk+in+patients+with+de+novo+acute+myeloid+leukemia:+a+report+from+Children's+Oncology+Group.">Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.</a> Blood. 120 (8), 1581-1588 (2012).</li><li>Zeijlemaker, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+one-tube+assay+for+immunophenotypical+quantification+of+leukemic+stem+cells+in+acute+myeloid+leukemia.">A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.</a> Leukemia. 30 (2), 439-446 (2015).</li><li>. Guidance for Collection, Transport and Submission of Specimens for Ebola Virus Testing Available from: <a target="_blank" href="https://www.cdc.gov/vhf/ebola/healthcare-us/laboratories/specimens.html">https://www.cdc.gov/vhf/ebola/healthcare-us/laboratories/specimens.html</a> (2017)</li><li>Bachas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+minor+subpopulations+within+the+leukemic+blast+compartment+of+AML+patients+at+initial+diagnosis+in+the+development+of+relapse.">The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.</a> Leukemia. 26 (6), 1313-1320 (2012).</li><li>Ravandi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+minimal+residual+disease+assessed+by+multiparameter+flow+cytometry+is+highly+prognostic+in+younger+patients+with+acute+myeloid+leukemia.">Persistence of minimal residual disease assessed by multiparameter flow cytometry is highly prognostic in younger patients with acute myeloid leukemia.</a> Cancer. 123 (3), 426-435 (2017).</li><li>Hourigan, C. 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The antibody cocktails used for the validation of the stem cell tube in several independent institutes were kindly provided by BD. The authors have nothing to disclose in relation to this manuscript.

Ample data are available that show evidence for MRD positivity being associated with poor survival, and therefore MRD assessment may improve patient outcome by providing additional prognostic information upon which clinical decisions can be made. Hence, a consistent and harmonized method for immuno-phenotypic assessment of MRD is essential to ultimately improve patient therapy. This is also important when comparing clinical studies across different clinical sites, and may ultimately help in clinical decision making and serving as a surrogate clinical endpoint for overall survival. The notion that solid guidelines are warranted for accurate and consistent MRD measurements has led to a concerted action of the European Leukemia Network (ELN) to design state of the art guidelines. This comprehensive document will be published late-2017, and will be instrumental for many study groups and laboratories moving forward. 
Critical steps within the protocol
An important, and often overlooked aspect of MRD analysis is the impact of sample quality on accurate determination of MRD. This is more apparent when material has to be transported to other institutes in global clinical trials given the additional operational considerations that need to be taken into account in this setting. To reduce the risk of mixing up patient information and timely delivery of the samples adequate administration is crucial. Again, at this stage of the transport the correct forms and/or import into electronic hospital systems with clear assignments of the requested analysis is required. Noting the stage at therapy of MRD sampling is also crucial since it may be relevant for clinical decision making (for example after second course of therapy) and should therefore be defined clearly. The use of mock data (such as for instance January 1 per year of birth) increases the risk of mixing up patients or analyses. If required by law, an alternative anonymized way of identification should be used.
All specimens for immunophenotyping should be processed preferentially within 24 h of collection. Although not recommendable, bone marrow and peripheral blood samples can still be processed and analyzed when kept up to 72 h at ambient temperature. In addition, all handlings with the material can best be performed under sterile conditions, so further cryopreservation of cells in (local) biobanks remains relevant: many clinical studies have additional analyses to further investigate leukemia cells with respect to molecular, immuno-phenotypic, and functional features to be performed at a later stage. However details of biobanking are not within the scope of this manuscript.
Using MRD as a diagnostic tool implies that it needs an accredited laboratory, not only for flow cytometry, but also regarding quality control of MRD assessment. This may require distributing samples to specific reference laboratories or (re)analyzing flow files with the MRD measurements by reference teams. 
This article describes the most important actions from bone marrow aspirate collection to determination of MRD in clinical samples - requiring an entire team of experts with specific tasks, responsibilities, and with frequent communication for effective characterization and analysis. Since each task is crucial to the procedure, it is recommended to have sound logistics including protocols at each laboratory and sufficient back-up personnel who have been specifically trained for the task. In addition, since there are some relatively subjective steps in part of the procedures (especially LAIP and LSC aberrant marker identification), it is essential to have discussions about the final result by the team, and have the final report authorized by a team of supervisors. Current efforts are pursuing computer software development that will help standardize the (LAIP and LSC) MRD analysis.
Modification and troubleshooting
Each lab can have its own set of antibodies that can be used to define the different subpopulations although having a standardized backbone of markers is essential for comparable results, as outlined in the ELN state of the art guidelines for MRD measurements document referred to above. Irrespective of the chosen markers there are some issues that can make the analysis of the results difficult and need to be taken into consideration. 
Limitations of the technique
The identification of LAIP and LSC aberrant marker expression is first assessed at diagnosis and monitored over time (during and after therapy) for accurate characterization of the MRD phenotype. While over 95% of the patients can be evaluated via LAIP or LSC (or both), still some patients have no defined LAIPs or no CD34+CD38- LSCs or present with CD34 negative blasts, or have a missing diagnosis sample. In these cases, it is still worthwhile to try and measure MRD with a panel of antibodies as broad as the number of available cells allows and then select the most reliable (giving the strongest distinction of leukemic cells) LAIP. Considering that a proportion of patients who do ultimately relapse do not completely resemble the diagnosis immunophenotype, due to the heterogeneity of AML disease, measuring a broad panel of antibodies at MRD is recommended anyhow. These immunophenotypic shifts include blast cells and LSCs and have been shown to occur during therapy16 and the measureable disease may be based on these so-called upcoming populations. At this time however, it has not been determined whether all immunophenotypic sub-populations will lead to disease relapse, and is therefore not common practice to report MRD tailored-therapy based on these cells in clinical trials. It is important to note that the risk of missing LSC due to population shifts is reduced by the one tube approach in which the most important aberrancy defining markers are in one flow cytometry (PE) channel14.
Significance with respect to existing methods
The method described in this protocol relies on the definition of one or more LAIPs, which cells are followed during therapy. A disadvantage of this method is that it has been recently shown that LAIP may change during therapy. This way some upcoming populations with different aberrant markers may be missed. To circumvent this, it would be best to measure all aberrant markers instead of only those found at diagnosis. This would then be similar to the "different from normal" approach that is used by several laboratories17.
Future applications
Recently, MRD has received extra attention for novel clinical trial design. In the era of specialized treatment options and targeted drug therapy, the use of MRD as an outcome measure will reduce the time needed to establish clinical efficacy of novel treatments, ultimately allowing faster introduction of urgently needed therapeutic options into the clinic. The significance of appropriate logistics and practical execution of a harmonized MRD assessment are crucial to future AML treatment success as the FDA is currently investigating the feasibility of using MRD as a surrogate endpoint instead of overall survival measures18.

Acute myeloid leukemia (AML) is a malignancy of the bone marrow characterized by defects in the maturation program, with abnormal proliferation and accumulation of myeloid progenitor cells, inhibition of normal hematopoiesis, and ultimately bone marrow failure. The disease is highly heterogeneous with respect to morphology, immunophenotype, cytogenetics, molecular aberrations, and gene expression signatures, as well as to treatment response and treatment outcome1,2. Current management includes induction chemotherapy with the aim to achieve complete remission (CR), followed by post-remission treatment, which is largely guided by the results of molecular, cytogenetic and immunophenotypic studies and consists of either several courses of additional chemotherapy or (autologous or allogeneic) stem cell transplantation3. Despite high remission rates after intensive chemotherapy of up to 90%, 5-year survival in adults is only approximately 30%-40%, predominantly due to the development of relapses which are commonly resistant to chemotherapy and thereby very difficult to treat. Outcome in children is better, although approximately one third also relapse. Therefore, early detection of imminent relapse will fill an unmet medical need and may guide post-remission therapy4. 
Residual disease after therapy may reflect the sum of all diagnosis and post-diagnosis resistance mechanisms/factors; hence its measurement may be prognostic and instrumental for guiding treatment. The possibility of de&#64257;ning residual disease (formerly called minimal residual disease and now referred to as measurable residual disease or MRD) far below the morphological criterion of 5% blast cells is changing the landscape of risk classi&#64257;cation. Currently the two methods mostly used to detect MRD are flow cytometry-based and molecular-based, the latter being assessed by reverse transcriptase PCR (RT-qPCR)5 or, although in a premature stage, by next generation sequencing (NGS). Many studies in adults as well as in children already demonstrated that various MRD approaches provide strong prognostic information in AML both after induction and consolidation therapy6,7, and a new definition of disease burden (superior to morphological CR) is now emerging8. This suggests that MRD assessed by flow and/or molecular techniques should become, and in fact are already becoming, standard in every clinical trial in AML. 
This manuscript discusses the detailed flow cytometry procedure to obtain an accurate and reproducible immunophenotypic characterization of MRD in bone marrow samples, including the bone marrow sampling and processing procedures preceding flow cytometry. The availability of good quality bone marrow samples at diagnosis and follow-up is crucial to the success of this measurement across clinical sites and clinical trials. In fact, these pre-analytical considerations are also of vital importance for molecular (PCR and NGS) MRD approaches. For immunophenotypic characterization of MRD, aberrantly expressed markers are combined with normal myeloid and progenitor markers, to identify a leukemia-associated immunophenotype (LAIP)9. MRD measures the resultant of many factors influencing response to therapy, such as intrinsic or acquired leukemia drug resistance, pharmacodynamics and kinetics of therapy, immune surveillance, and compliance. Therefore, MRD is a very strong post-diagnosis prognostic parameter associated with clinical outcome when dichotomized on a cut-off level determined by Receiver-Operating Characteristic (ROC) analysis. For our adult AML cohort of the HOVON 42a study, the cut-off level is set at 0.1% of LAIP positive cells/total white blood cells. Using this criterion for determining negative vs positive MRD status, a group of patients can be identified who have a significantly worse relapse incidence, relapse-free and overall survival6. Additionally, we describe the measurement of immature, drug resistant leukemia cells with stem cell-like features (CD34+CD38- leukemia stem cells, or LSC), which offers a strong predictor of patient outcome10. Together LAIP and LSC approaches form the flow cytometric MRD approach. The LAIP approach is suitable for roughly 90% of the patients, while the LSC approach can be applied in about 80% of the patients. Together over 95% of the patients can be evaluated for either one parameter or both. 
Lastly, this publication provides a detailed operational description to assess MRD by flow cytometry. This includes: 1) harmonization and/or standardization of bone marrow sampling procedures, 2) sample transportation guidance 3) detailed description of the leukemic cell detection with FACS using several antibody panels including single cell tube approaches to characterize the LSC, 4) set-up of the FACS machines for standardized measurements, 5) analytical programs for MRD measurements and 6) analytical programs for LSC detection.
We aim to show all facets of the procedure including the sample preparation since that is rarely discussed while it is an important issue for the quality of the ultimate result. Bone marrow aspiration and biopsy are clinical procedures used to evaluate the hematopoietic cells within bone marrow. These are performed together with a complete blood count (CBC) and blood smear. The optimal method for bone marrow aspiration is crucial for the accurate diagnosis and follow-up for MRD measurement. In addition, a successful bone marrow aspirate should contain enough cells to perform the LAIP and LSC flow analysis (at least 10 million viable cells). Here, we describe the method for performing a bone marrow aspiration and provide guidelines, which should result in adequate cell sampling (and limit the potential for hemodilution) needed for accurate diagnostics and additional research. These pre-analytical considerations are also of vital importance for molecular (PCR and NGS) MRD approaches. All specimens for immunophenotyping should be processed preferentially within 24 h of collection. Although not recommendable, bone marrow and peripheral blood samples can still be processed and analyzed when kept up to 72 h at ambient temperature. In addition, all handlings with the material should be performed under sterile conditions, in order to enable cryopreservation of sterile cells for later research/quality assessment etc.

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comprehensive protocol to sample and process bone marrow for measuring measurable residual disease and leukemic stem cells in acute myeloid leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

In 90% of AML patients at least one LAIP can be identified. In order to generate the accurate percentages of LAIP+ cells of the primitive blast cells at diagnosis, the appropriate setting of the blast gate is crucial and needs verification as visualized in Figure 3. An example of each patient harboring a specific type of aberrancy on the CD34+ primitive cells is visualized in Figure 5. For measurement of MRD at follow-up,the earlier defined LAIP+ cells are gated and defined as percentage of LAIP+ cells of the WBC. Hence the setting of this gate is crucial to achieve the right MRD results. Figure 6 shows a patient who remains in complete remission and a patient that relapses after having MRD positive results (&#8805; 0.1 %) after the second cycle of induction therapy.
The current protocol is only suitable to define stem cells in CD34+ cells, since thorough characterization of this compartment has shown that the CD38- population harbors the most potent stem cell like fraction. In order to separate the LSC from the HSC within this fraction, additional markers are used. Most often selected markers to distinguish LSC are CD45RA and the combi channel harboring 6 specific LSC markers labeled with PE. Based on further clinical evaluations, a cut-off level of &#8805; 0.004% was defined as LSC-positive and was associated with worse survival at diagnosis while a cut-off level of 0.0001% was used at follw-up10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56386/56386fig1.jpg" /> 
Figure 1: Bone marrow smear. A) healthy donor and B) AML patient (FAB 5 subtype). Giemsa stain shown at 100x magnification. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56386/56386fig2.jpg" /> 
Figure 2: Correct packaging material for biological fluids. More details on the rules and regulations of transporting biological fluids can be found at the website15 of Centers for Disease Control and Prevention. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56386/56386fig3.jpg" /> 
Figure 3: Gating strategy for defining blast cells. A) Gating CD45dim, B) Gating CD34+ blasts.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56386/56386fig4.jpg" /> 
Figure 4: Gating lymphocytes. A) Blue cells are WBC shown in FSC/SSC plot, B) Green cells are lymphocytes characterized by CD45high/SSClow, C) Examples of negativity for CD34, D) negativity for CD117 and E) negativity for CD13, F) The different cell types are shown in an overview population tree. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56386/56386fig5.jpg" /> 
Figure 5: LAIPs at AML diagnosis. A) LAIP based on cross lineage aberrancy (CD7 on CD33 expressing cells), B) LAIP based on asynchronous differentiation (CD11b on CD13 expressing cells), C) LAIP based on overexpression compared to normal cells (CD34very high on CD13 expressing cells), D) LAIP based on underexpression compared to normal cells (HLA-DRlow on CD33 expressing cells). <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56386/56386fig6.jpg" /> 
Figure 6: LAIP followed during therapy. A) LAIP definition at diagnosis (CD34+/CD13+/CD33-) and loss of LAIP during follow-up in a patient who remained in continuous complete remission, B) LAIP definition at diagnosis (CD117+/CD7+/CD33+) and persistence of LAIP during follow-up in a patient who relapsed. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56386/56386fig7.jpg" /> 
Figure 7: Stem cell gating. A) Gating of the different CD34+/CD38 populations based on beads (CD38low is below the upper border of the beads, while CD38very low are defined as the true negative cells representing the LSC and are located below the median of the beads), B) two examples of patients with distinction between HSC and LSC at follow-up based on CD45RAneg and CD45RApos expression. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/56386fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/Sampling_Form_MRD_1.pdf" target="_blank">Please click here to downoad this file.</a>
Supplemental File 2. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/Sampling_Form_MRD_2.pdf" target="_blank">Please click here to downoad this file.</a>
Supplemental File 3. <a href="https://cloudflare.jove.com/files/ftp_upload/56386/Supplementary_information_revised_Cloos_et_al.zip" target="_blank">Please click here to downoad this file.</a>

Watch this Scientific Journal Video about Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia at JoVE.c

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

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