|Year : 2016 | Volume
| Issue : 1 | Page : 17-23
Minimal residual disease program for acute lymphoblastic leukemia at Dhahran Health Center
Nasir Khalid Amra1, Salwa Shaberdeen Sheikh1, Basel A Abushullaih2, Nafeesa A Al-Faris2, Adil A Al-Khatti2, Huda H Al-Sayed3
1 Pathology Services Division, Johns Hopkins Aramco Healthcare, Dhahran 31311, Saudi Arabia
2 Hematology/Oncology Services Division, Johns Hopkins Aramco Healthcare, Dhahran 31311, Saudi Arabia
3 Clinical Laboratory Services Division, Johns Hopkins Aramco Healthcare, Dhahran 31311, Saudi Arabia
|Date of Web Publication||25-Apr-2016|
Nasir Khalid Amra
Dhahran Health Center, John Hopkins Aramco Healthcare, Building 62, Room No. 294, Dhahran 31311
Source of Support: None, Conflict of Interest: None
Background and Objectives: Minimal residual disease (MRD) assays for monitoring acute lymphoblastic leukemia (ALL) during treatment are defined as assays with a limit of detection of at least 0.01% leukemic blasts per mononuclear cells or total nucleated cells. Settings and Design: We retrospectively reviewed out experience at Dhahran Health Center in monitoring adult and pediatric ALL patients with a MRD assay based on immunophenotyping by flow cytometry with a level of detection of 0.01% leukemic blasts per mononuclear cells and compute Kaplan–Meier survival analysis for overall survival (OS) and relapse-free survival (RFS). We also demonstrated the incorporation of an estimated measurement uncertainty for the reported MRD values based on metrological principles. Methods: A retrospective review of all cases diagnosed with ALL from 2006 to 2012 was undertaken and after applying exclusion criteria, 26 cases were identified and patient chart review was done. Results: Although the Kaplan–Meier survival analysis for OS and RFS do demonstrate a statistically significant difference between MRD positive and negative patients, none of the pediatric ALL MRD positive cases have relapsed till now. Conclusions: The detection of MRD in ALL opens up the opportunity to intensify or alter treatment for patients with detectable levels by a highly sensitive assay before clinical relapse.
Keywords: Acute lymphoblastic disease, flow cytometry, minimal residual disease
|How to cite this article:|
Amra NK, Sheikh SS, Abushullaih BA, Al-Faris NA, Al-Khatti AA, Al-Sayed HH. Minimal residual disease program for acute lymphoblastic leukemia at Dhahran Health Center. J Appl Hematol 2016;7:17-23
|How to cite this URL:|
Amra NK, Sheikh SS, Abushullaih BA, Al-Faris NA, Al-Khatti AA, Al-Sayed HH. Minimal residual disease program for acute lymphoblastic leukemia at Dhahran Health Center. J Appl Hematol [serial online] 2016 [cited 2020 Apr 9];7:17-23. Available from: http://www.jahjournal.org/text.asp?2016/7/1/17/181113
| Introduction|| |
With the success of modern chemotherapy regimes in curing pediatric acute lymphoblastic leukemia (ALL), research in this area has expanded to looking at new prognostic markers to stratify patients during chemotherapy to alter chemotherapy regimens either to reduce intensity of chemotherapy when not needed or to increase intensity of chemotherapy for those identified to be at high risk of relapse. Past seminal studies ,,, have demonstrated that using immunophenotyping by flow cytometry to quantitate the leukemic blast burden at particular time points during chemotherapy to a level of detection of one leukemic blast in 10,000 mononuclear cells could be used for prognostic stratification of risk for early relapse. In contrast, the morphologic assessment of the bone marrow aspirate has a level of detection of 5 leukemic blasts per 100 cells.
Dhahran Health Center, to the best of our knowledge, was the first institution in Saudi Arabia to initiate and sustain this complex immunophenotyping flow cytometry assay for minimal residual disease (MRD) in ALL. We retrospectively performed a clinical audit of this assay and summarized our experience in implementing a MRD assay with a level of detection of 0.01% blasts per mononuclear cells by flow cytometry immunophenotyping for ALL. We also illustrated how applying the principles of metrology, one can calculate an estimated 95% confidence interval (CI) for MRD assay results.
| Methods|| |
A retrospective review of all cases diagnosed with acute lymphoblastic leukemia from 2006 to 2012 was undertaken. Cases of Burkitt leukemia/lymphoma, bilineal (2 distinct neoplastic cell populations) leukemia, leukemia with t(9;22) chromosomal translocation, and death during remission induction were excluded from the study. Twenty-six cases were identified. At diagnosis, nearly all patients had conventional cytogenetic studies, DNA analysis by flow cytometry, and FISH studies for the common translocations in ALL. All patients had subsequent postremission induction bone marrow samples (most cases at day 29 with two cases delayed till day 31 and day 37) that were assayed for MRD by flow cytometry immunophenotyping. Some patients also had the MRD assay done at other time points during treatment either during remission induction or during consolidation. The pediatric patients (<18 years of age) were treated as per different Children's Oncology Group treatment regimens. Of the seven adult patients (>18 years of age), 5 were also treated per Children's Oncology Group treatment regimens for high-risk patients, one was treated with a MD Anderson cancer center protocol of hyper-cyclophosphamide, vincristine, doxorubicin, and dexamethasone/methotrexate-cytarabine, and another adult patient was treated but medical records were not available for review to abstract the details of the treatment plan. This study was approved by the Dhahran Health Center Institutional Review Board and informed consent was obtained from all subjects or their guardians.
Flow Cytometry Method
At Dhahran Health Center, MRD assays for B- or T-cell precursor ALL to look for a leukemia aberrant immunophenotype pattern (LAIP) is performed during the assessment of the diagnostic bone marrow aspirate utilizing a panel of fluorescent antibodies derived from the prior studies., The samples are incubated with the reagents and processed using the “stain/lyse/wash” method for staining of the surface antigens on a four-color Becton Dickenson (BD) FACSCaliber flow cytometer using CellQuest Pro analysis software or BD FACSCanto II flow cytometer using FACSDiva analysis software (BD Biosciences, San Jose, California, USA). FIX and PERM Permeabilization Kit (Caltag Laboratories, Burlingame, CA, USA) was used according to manufacturer's instructions for examination of intracellular antigens. Monoclonal antibodies used for MRD monitoring were purchased from the following manufacturers: BD Biosciences reagents include anti-IgM fluorescein isothiocyanate (FITC), CD235a-phycoerythrin (PE), CD45-peridin chlorophyll protein (PerCP), CD45-allophycocyanin (APC), CD61-FITC, CD33-APC, CD10-PE, CD19-PerCP, CD20-FITC, CD34-PE, HLA-DR-PerCP, CD117-PE, CD13-PE, CD34-PerCP, CD9-FITC, CD56-PE, CD11c-PE, CD19-APC, CD34-FITC, CD4-PE, CD8-PerCP, CD15-FITC, CD22-FITC, CD33-FITC, CD45-FITC, CD58-FITC and CD65-FITC for surface antigens; IOTest Beckman Coulter reagents include CD11b-FITC, CD7-PE, CD21-FITC, CD38-FITC, CD10-FITC, 7.1 (anti-NG2)-PE and CD66 (Korsa3544)-FITC for surface antigens; BD Biosciences intracytoplasmic reagents include CD3-FITC, CD3-PerCP, CD79a-PerCP, CD22-APC; IOTest Beckman Coulter MPO-FITC is used for detecting intracytoplasmic antigen; IOTest Beckman Coulter is used for TDT-FITC nuclear antigen detection.
At diagnosis, the bone marrow aspirate was analyzed utilizing a screening panel of antibodies for leukemia lineage classification (myeloid, B-cell, or T-cell). Subsequently, a more extensive panel was used to identify one or more LAIPs to monitor the patient's LAIP status during chemotherapy on subsequent bone marrow or peripheral blood samples. For precursor B-cell acute leukemia, the panel consists of TDT/CD10/CD34/CD19, CD15/CD10/CD34/CD19, CD21/CD10/CD34/CD19, CD22/CD10/CD34/CD19, CD33/CD10/CD34/CD19, CD38/CD10/CD34/CD19, CD45/CD10/CD34/CD19, CD58/CD10/CD34/CD19, CD65/CD10/CD34/C19, CD66c/CD10/CD34/CD19, IgM/CD10/CD34/CD19, CD10/CD13/CD34/CD19, CD10/56/CD34/CD19, CD10/NG2/CD34/CD19 and TDT/Cyto-IgM/CD34/CD19. For precursor T-cell acute leukemia, the panel consists of TDT/CD7/Cyto-CD3/CD19 + CD33, CD34/CD7/Cyto-CD3/CD19 + CD33, CD3/CD4/CD8/CD19 + CD33, and CD3/CD5/CD8/ CD19 + CD33.
One or more LAIP was identified consisting either coexpression of antigens from different cell lines or asynchronous, overexpression, or underexpression of antigens within the same cell line.,
The postremission induction bone marrow samples were assayed for MRD using three different tubes of fluorochrome conjugated antibody or dye reagents. For example, the LAIP at the time of diagnosis may have an aberrant expression of CD58 on CD19, CD10, and CD34 positive B-cells. Subsequent analysis for MRD would use three different tubes of reagents ( Syto16, CD45 PerCP, and CD19 APC;  CD19 APC, CD34 PerCP, CD10 FITC, and CD58 PE;  CD19 APC, CD34 PerCP, CD10 FITC, and mouse IgG1 PE) to quantify the LAIP blast burden. The bone marrow sample is washed 3 times with BD cell wash, and then initially incubated with negative control immunoglobulin (DakoCytomation) to block FC receptors. Subsequently, the sample is washed and then incubated with the above three tubes of reagents. After incubation, the tubes are run through the flow cytometer and analyzed with the BD CellQuest Pro or FACSDiva software.
[Table 1] enumerates what data are extracted from each tube and gives a variable name for each event type. (See [Figure 1] for sample dotplots of LAIP determination). From tube 1, the fraction of B-cells within the viable mononuclear cells is t/m. From tube 2, the fraction of LAIP positive blasts within the B-cell population is L/s. From tube 3, the fraction of background (noise) events within the same region as LAIP positive blasts within the B-cell population is n/b. This then allows us to compute the fraction of LAIP positive blasts within the viable mononuclear population as shown in Equation 1. When the calculated result of Equation 1 is ≥0.0001, the case is considered MRD positive.
|Table 1: Measurements extracted from experimental run of minimal residual disease for precursor B-cell ALL using CD19+/CD34+/CD10+/CD58+ leukemia associated immunophenotype as an example|
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|Figure 1: Dotplots illustrating sequential selective gating on day 8 bone marrow aspirate on a sample bone marrow of a patient with acute lymphoblastic leukemia demonstrating a leukemia aberrant immunophenotype pattern consisting of CD19+/CD10+/CD34+/CD58 + leukemic blasts. The top row represents tube 1 which generates t/m (Equation 1). Note that dotplots (c) and (d) are gated on regions P1 of (a) and P2 of (b). The middle row represents tube 2 which generates L/s. Note that dotplot (f) is gated on P1 of (e) while dotplot (g) is gated on regions P1 of (e) and P6 of (f). Dotplot (h) has region P5 that represents the region of leukemic blasts with leukemia aberrant immunophenotype pattern where the gate is a conjunction of regions P1 of (e), P6 of (f), and P3 of (g). The bottom row represents tube 3 which generates n/b and provides a measure of noise in the leukemia aberrant immunophenotype pattern region. Using a fluorescent minus one isotype control with similar sequential gating (dotplots i–l) as in the dotplots in the middle row|
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The fraction of targeted cells in a mononuclear cell population can be modeled as a binomial experiment to compute a proportion P = t/c where t and c are arbitrary numbers and the variance of P is computed as p*(1 − p)/c. The estimated variance for Equation 1 can be calculated by error propagation rules  to be Equation 2 [Supplement A for details [Additional file 1]]. This variance can be used to construct an estimated 95% CI for the MRD assay result by taking its standard deviation to get the measurement uncertainty. Multiplying the measurement uncertainty (standard deviation) by 1.96 and adding this to the MRD assay result will give the upper bound of 95% CI. Similarly, multiplying the measurement uncertainty (standard deviation) by 1.96 and subtracting this from the MRD assay result will give the lower bound of 95% CI.
[Table 2] shows the measurements and computations on a sample patient using Equation 1.
Analysis and Statistical Methods
Overall survival (OS) is defined as time from diagnosis (with start of treatment that day) to death or last follow-up. Relapse-free survival (RFS) is only determined for patients that achieved remission induction and is defined as the time from start of diagnosis to relapse or last follow-up. RFS and OS were estimated using the methods of Kaplan and Meier. Differences between survival curves were assessed for statistical significance using the log-rank statistics. The pediatric population is defined as <18 years of age.
| Results|| |
Since 2006, the Dhahran Health Center has initiated and sustained a program to do MRD assay for ALL by flow cytometry immunophenotyping to support our hematologists in following the Children Oncology Group ALL treatment protocols for pediatric patients. The MRD assay was also used for risk assessment on adult ALL patients. In the period between 2006 and 2012, 26 ALL cases were included in this study comprising 5 T-cell ALL, 1 T-cell/myeloid biphenotypic leukemia, and 20 precursor B-cell ALL cases. The characteristics of the patients included in this study are listed in [Table 3]. [Table 4] lists the pattern of LAIPs identified in this set of patients at diagnosis. Eight cases of the B-lineage ALL had 2 LAIPs identified per case which comprise 40% of the B lineage cases. All ALL cases had at least one LAIP. Of the 20 precursor B-cell ALL patients, 6 were positive for MRD at postinduction chemotherapy. Two of the positive MRD adult ALL cases subsequently relapsed and died of disease. One of the relapsed adult patients had a central nervous system and bone marrow relapse, but with a different immunophenotype from the LAIP identified at diagnosis (from CD38 negative at diagnosis to CD38 positive and increased intensity of CD10 expression in both CSF and bone marrow relapse samples).
Of the remaining 15 precursor B-cell ALL cases that were MRD negative, none had relapsed. Of the 5 T-cell ALL cases, 2 were positive for MRD at postremission induction time point. None of the 5 T-cell ALL cases have relapsed or died from disease up till now. The T-cell/myeloid biphenotypic leukemia was negative for MRD and still alive without disease. The Kaplan–Meier survival analysis for OS and RFS do demonstrate a statistically significant difference between MRD positive and negative patients [Figure 2] and [Figure 3]. None of the pediatric ALL patients (19 cases) with positive MRD results (4 cases of precursor B-cell ALL and 1 case of T-cell ALL that have MRD results ranging from 0.01% to 0.08% LAIP blasts per mononuclear cells) have relapsed.
|Figure 2: Overall survival Kaplan–Meier analysis stratified by minimal residual disease status|
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|Figure 3: Relapse-free survival Kaplan–Meier analysis stratified by minimal residual disease status|
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| Discussion|| |
ALL represents 80% of pediatric acute leukemia and 20% of adult acute leukemia. In the USA, the age-adjusted incidence of ALL is 1.6/100,000 men and women. However, in Saudi Arabia, the age-adjusted incidence of ALL is 3.3/100,000 men and women. With modern chemotherapy protocols, childhood ALL can achieve a cure rate of 90% while adult ALL can achieve a cure rate of 30–40%.
Recently, clinical trials for the treatment of ALL in Europe and USA have not only assessed standard prognostic risk factors in ALL (age, white blood cell count, DNA index, and specific chromosomal translocations) but also incorporated a MRD assay (defined as an assay that has a level of detection of at least 0.01% leukemic blasts per mononuclear cells or total nucleated cells), that is, performed at various time points during chemotherapy on either bone marrow or peripheral blood samples. The MRD assay is implemented either by detecting leukemia-associated immunophenotype patterns by flow cytometry or polymerase chain reaction assays targeted at either patient specific immunoglobulin, T-cell receptor gene rearrangements, or leukemia-associated fusion genes. Numerous studies have demonstrated the independent prognostic significance of a positive MRD result in ALL as a risk factor for relapse in both adults and children.,,,,15],,,, The concordance between molecular and flow cytometry MRD assays is between 89% and 91.3% at a cutoff detection level of 0.01%.
Although this study is limited by its size and follow-up of the patients, it does demonstrate the prognostic significance of MRD status in ALL patients for RFS and OS. Additional therapeutic interventions were taken for some of the positive MRD patients. In one case, an adult T-cell ALL MRD positive patient had consolidation started without waiting for peripheral blood recovery with subsequent delayed intensification. The second MRD assay after the first interim maintenance was negative and the patient ultimately underwent bone marrow transplantation. The relapsed 20-year-old female B-cell ALL case was started on an adult B-cell ALL protocol. Her day 29 MRD result was 1.7% and she continued treatment on a pediatric ALL protocol until development of a cerebrospinal fluid and bone marrow relapse 28 months after start of chemotherapy. She subsequently underwent bone marrow transplantation and died of her disease. A recent meta-analysis comparing adolescents and young adults (16–39 year-old) with ALL demonstrated a reduction in all-cause mortality in patients given pediatric-inspired regimens compared with patients given conventional adult regimens (relative risks 0.59; 95% CI 0.52–0.66) although it is not clear if patients older than 20 years benefit from pediatric-inspired regimens.
Of the 5 MRD positive pediatric ALL cases, 2 cases were below 0.1% and started treatment before 2008 when the threshold for changing chemotherapy regimen was an MRD positive value of 0.1% or more. Two of the remaining 3 cases were considered high risk based on traditional high-risk parameters (white blood cell count, patient age, or T-cell subtype of ALL). The last case had a MRD positive value of 0.01% and continued his low-risk treatment protocol.
A recent review has emphasized the importance of MRD assay measurements in prognostication and initial evidence as to the benefit of altering therapy of ALL in response to a positive MRD result. Another review points to future enhancement of level of detection of MRD assays using a variety of techniques and technologies. A sensitive MRD assay incorporated as an early biomarker of response to therapy during treatment of ALL will not only open the door to better prognostication of the likely course of the individual patient but also select candidates for clinical trials with innovative therapeutic agents before frank relapse when the disease burden is relatively low.
| Conclusion|| |
The detection of MRD in ALL with a highly sensitive assay of at least 0.01% provides the opportunity intensify or alter treatment of patients before clinical relapse.
We acknowledge the use of John Hopkins Aramco Healthcare (JHAH) facilities for the data utilized in this manuscript. Opinions expressed in this article are those of the authors and not necessarily of JHAH. We would also like to acknowledge the dedication and support of Ms. Reem Qattan and Zainab Sawad, flow cytometrists at Dhahran Health Center.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]