Detection of Minimal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight-Color Tube with Dried Antibody Reagents

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Detection of Minimal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight-Color Tube

with Dried Antibody Reagents

Lakhdar Bouriche, Denis Bernot, Vanessa Nivaggioni, Isabelle Arnoux, Marie Loosveld

To cite this version:

Lakhdar Bouriche, Denis Bernot, Vanessa Nivaggioni, Isabelle Arnoux, Marie Loosveld. Detection

of Minimal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight-Color Tube

with Dried Antibody Reagents. CYTOMETRY PART B-CLINICAL CYTOMETRY, 2019, 96 (2),

pp.158-163. �10.1002/cyto.b.21766�. �hal-02359455�

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Brief Communication

Detection of Minimal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight-Color Tube

with Dried Antibody Reagents

Lakhdar Bouriche,¹Denis Bernot,¹Vanessa Nivaggioni,¹Isabelle Arnoux,¹and Marie Loosveld^1,2*

1Assistance Publique Hôpitaux de Marseille, Laboratoire d’Hématologie, Hôpital de la Timone, Marseille, France

2CNRS, INSERM, CIML, Aix Marseille University, Marseille, France

Background: Flow cytometry is a powerful tool for the detection of minimal residual disease (MRD) of B cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. However, the staining process and the choice of antibodies rely on laboratory expertise and may be source of variability or technical errors. Recently, Beckman Coulter commercialized a ready to use tube with dried format reagents for BCP-ALL MRD detection. The aim of this study is to evaluate the applicability of this tube and to compare it to a conventional (liquid format reagents) method.

Methods: Thirty-one samples from B ALL patients were analyzed: 19 bone marrow (BM) aspirations, 10 peripheral blood (PB) samples and 2 cerebrospinalﬂuids at different stages of the follow-up. In addition, we tested 5 bone marrow samples mixed into non-pathological (control) bone marrow. The dried format tube included seven antibodies: CD45Kro, CD58FITC, CD34ECD, CD10PC5.5, CD19PC7, CD38AA700, CD20AA750, with possibility of additional antibodies for blast markers identiﬁed at diagnosis. For comparison, a liquid format tube was prepared, and considered as the reference.

Results: This tube was validated for daily routine laboratory, with satisfying qualitative (MRD + or MRD−) and quantitative (MRD percentages) correlation with the reference tube.

Conclusion: With this single dried format tube, we showed interesting results for BCP-ALL MRD detection in the aim of standardization and reliable interlaboratory results. It allows accurate MRD detection including low levels (10–4), and offers possibility to increase performance (supplementary antibody) within a preestablished effective antibody panel for BCP-ALL MRD.© 2019 International Clinical Cytometry Society

Key terms: ﬂow cytometry; B cell acute lymphoblastic leukemia; minimal residual disease detection; Dura- clone; dried reagents.

How to cite this article:Bouriche L, Bernot D, Nivaggioni V, Arnoux I, and Loosveld M. Detection of Mini- mal Residual Disease in B Cell Acute Lymphoblastic Leukemia Using an Eight-Color Tube with Dried Anti- body Reagents. Cytometry Part B 2019; 96B: 158–163.

Minimal residual disease (MRD) assessment repre- sents a predictive and independent risk factor and a useful tool in the clinical management of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) (1,2).

Two methods coexist for studying MRD: Multipara- metric Flow Cytometric (MFC) analysis of leukemia- associated immunophenotypes (LAIP) and polymerase chain reaction (PCR) ampliﬁcation of antigen receptor rearrangements (3). Garand et al. have demonstrated in a multicenter study that PCR and MFC are highly com- plementary strategies for MRD detection in ALL (4) and several studies have shown that the use of multicolor (>6 markers) immunostainings combined with the

introduction of speciﬁc newly described markers signif- icantly improved FCM-MRD analysis in BCP-ALL (5–8).

Among the advantages of the FCM, the possibility to have results just a few hours after sample collection is highly appreciated by clinicians, especially for patients who need a speciﬁc care.

*Correspondence to: Dr Marie Loosveld, 264 rue Saint Pierre, 13005 Marseille, France. Email: marie.loosveld@ap-hm.fr

Received 8 June 2018; Revised 13 December 2018; Accepted 9 January 2019

Published online 30 January 2019 in Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/cyto.b.21766

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Nevertheless, the workflow in clinicalflow cytometry laboratories must constantly be reviewed to develop technical procedures that improve quality and produc- tivity, and reduce costs. Indeed, clinical flow cytometry laboratories face discrepancies due to pipetting errors, short shelf life of products, planification of cocktail reagents, lot to lot potential variation, and some lack of standardization.

Classically (and it is the case for our laboratory), a combination of 8 to 10-color tubes is used to analyze MRD at different time points (peripheral blood or bone marrow MRD analysis). In this context, Beckman Coulter^® recently introduced a range of dried reagents with a speciﬁc tube for the detection of B Acute Lym- phoblastic Leukemia MRD called “Duraclone^® Rare Events ALB”(RE-ALB).

The aim of this study is to evaluate the applicability of the RE-ALB tube in routine laboratory and test performances of this dried format reagent when compared to the currently used liquid format reagent cocktail (control tube).

MATERIALS AND METHODS Patients and Samples

Twenty-two children (15 girls and 6 boys) with a de novo diagnosis of B-lineage ALL were considered for enrollment. Informed consent was obtained from par- ents/guardians and study protocols were approved by the ethics committee of Marseille University Hospital.

Samples from these patients were analyzed by morpho- logical examination and ﬂow cytometry in our laboratory.

Overall, 31 samples from B ALL patients were analyzed: 19 bone marrow (BM) aspirations, 10 peripheral blood (PB) samples and 2 cerebrospinalﬂuids (CSF) at different stages of the follow-up: during the corticotherapy (n= 11), at the end of induction (MRD1;n= 8), at the end of consolidation (MRD2; n = 6), at relapse (n= 3), and at other time points (OTP;n= 3). In addition, ﬁve bone marrow samples were diluted into a non-pathological bone marrow. Finally, samples from patients with cytopenia but without any malignant cells were used as control samples. These control subjects matched on age with pediatric patients.

Patients and samples characteristics are summarized in Table 1.

Sample Preparation

PB and BM samples were processed forﬂow cytometry analysis within 24 h after collection. In order to identify MRD at low level (10⁻³ or 10⁻⁴), we must enrich the sample by performing erythrocyte prelysis on a maximal number of cells before staining. Red blood cells were ﬁrst lysed using NH4Cl solution and the remaining cells were sequentially centrifuged (600 G) for 10 min, washed twice in phosphate buff- ered saline (PBS: pH 7.2) and resuspended in PBS. PB

and BM samples were diluted in order to obtain 10⁶ cells in 100 μl. Secondly, cells were incubated in the dark at room temperature for 15 min with the antibody cocktail. On the other hand, CSF samples were directly incubated with the cocktail without lysing and washing steps.

The liquid reagent tube was tested in parallel and considered as the reference. This tube included seven ﬂuorochrome-conjugated liquid antibodies with CD45Krome Orange (J.33), CD10FITC (ALB1), CD34 ECD (581), CD58PC5 (AICD58), CD19PC7 (J3-119), CD38APC (HB-7), CD20 AA750 (B9.E9 [HRC20]).

The Duraclone^®tube included seven Beckman Coulter ﬂuorochrome-conjugated dried antibodies: CD45Krome Orange (J.33), CD58FITC (AICD58), CD34ECD (581), CD10PC5.5 (ALB1), CD19PC7 (J3-119), CD38 AA700 (LS198-4-3), CD20 AA750 (B9.E9 [HRC20]).

FL2 channel was used to add a blast-speciﬁc marker (LAIP) antibody conjugated with PE, which was the same for liquid or dried reagent experiments. As a result, each tube contained eight antibodies. Data were immediately acquired.

Flow Cytometer and Quality Control

The instrument used was a Navios (Beckman Coulter^®, Miami, FL). Daily controls were run, including Flow Check and Flow Set Pro Fluorospheres™ (Beckman Coulter) to monitor the instrument performance and Immuno-Trol™ Cells as part of the electronic Interna- tional Quality Assurance Program (www.beckmancoulter.

com). As theﬂuorochromes differed between the two test tubes, different settings and a different compensation matrix were used for the Duraclone^®tube.

Compensations were made using dried-antibody coated single color tubes of the Compensation Kit pro- vided for Duraclone tests, according to manufacturer’s instructions.

Analysis Data

Analysis of ﬂow cytometry results was made using Kaluza software (Beckman Coulter). As usually in FCM- MRD analysis, we did not analyze MFIs of each isolated marker but studied correlation between the two staining methods on the obtained MRD quantitation’s. Three observers analyzed each MRD positive and negative case. Results were conﬁrmed by double blinded analyses.

Of note, because several tandem dye are used in the two format (dry and liquid tubes), the stability of these tandem dye (PC5, AA700…) are periodically controlled by searching a positive interference-signal in the poten- tially affected channel (PE or APC channel) depending on the considered tandem.

Statistical Evaluation

Results have been evaluated both qualitatively and quantitatively.

B-ALL MRD ASSESSMENT WITH MULTICOLOR DRY TUBE 159

Cytometry Part B: Clinical Cytometry

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Table1 Patientsandsamplescharacteristics Patients characteristicsSamples characteristics SexAge (months)

Leucocytes count (x109/L)Hemoglobin level(g/L) Platelets count (x109/L)

PB blasts (%)EGIL

Cytogenetic and/or molecular biologyResponseto corticotherapy Endof induction MRD byCMFPrognostic evaluation Number ofsamples analyzedTime Point

Type of samples Patient1F1804912115BIIBOTHERCoNE>10−3 CAALLVHR1OTPMO Patient2M486754450BIIHyperdiploidCoS<10−3FRALLEA11MRD2MO Patient3F24259812348BIIHyperdiploidCoS<10−3 CAALLSR1MRD2MO Patient4F180261102358BIILiFraumeni +IKZF1CoS>10−3CAALLHR2MRD1+ MRD2MO Patient5F2454861384BIIIBOTHERCoR<10−3CAALLHR1D1MO Patient6M4868111620BIIHyperdiploidCoS>10−3 CAALLHR2MRD2+ OTPMO Patient7M204161120BIIHyperdiploid +IKZF1CoNE>10−3 CAALLHR1D8PB Patient8F3625798859BIIETV6/ RUNX1CoS<10−3 CAALLSR1D4PB Patient9F2465632081BIIETV6/ RUNX1CoS<10−3 CAALLMR1MRD1MO Patient10F4833551773BIIETV6/ RUNX1CoS<10−3 CAALLSR2MRD1+ MRD2MO Patient11F962923250BIIHyperdiploidCoNE<10−3 CAALLSR1MRD1MO Patient12F4835719285BIIETV6/ RUNX1CoS<10−3FRALLEA11RelapseCSF Patient13F24729364BIIHyperdiploidCoS<10−3CAALLSR1D8PB Patient14F8421046412BIIHyperdiploidCoS<10−3 FRALLEA11RelapseMO Patient15F108340744187BIIPhi+IKZF1CoR<10−3EsPhALL3D8+ MRD1+ OTP

PB+MO Patient16F13241043289BIINearhaploidCoNE>10−3 CAALLHR2D1+D4PB Patient17F7219893378BIIBOTHERCoS<10−3FRALLEA11RelapseCSF Patient18F1462565210BIIHyperdiploidCoNE<10−3 CAALLMR1MRD1MO Patient19M23243653799BIIHyperdiploidCoR>10−3FRALLEB22MRD1+ MRD2MO Patient20M44717810491BIIBOTHERCoR<10−3CAALLMR1MRD1MO Patient21M357.5866150BIIHyperdiploidCoSNDCAALL2D4+D8PB Patient22M1252.4571130BIIPhiCoNENDEsPhALL2D4+D8PB CoS=Corticosensitive;CoR=Corticoresistant;CoNE=Cortico-non-evaluable;D1=Day1ofthecorticotherapy;D4=Day4ofthecorticotherapy;D8=Day8ofthe corticotherapy;MRD1=EvaluationoftheMRDattheendoftheinduction;MRD2=attheendoftheconsolidation;OTP=othertimepoint;ND=Notyetdetermined

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In the quantitative comparison, the two methods of FCM data were evaluated using nonparametric Spearman correlation statistics. All calculations were performed with Prism GraphPad Software^®.

RESULTS Applicability of the RE-ALB tube

Several control bone marrow samples were ﬁrst run with the Duraclone tube for “backbone marker” dried antibody validation and to test the reliability of this method for B lymphoid cells analysis (example: Fig. 1A).

When compared to counterpart liquid tube, the different cellular populations were positioned as expected, nota- bly on the SS versus CD45 cytogram. The same number of cells was analyzed, (approximatively 1 million) and

the same percentage of B lymphocytes was observed for both approaches (12%). Moreover, no cluster or signiﬁ- cant number of cells could be observed in the “blast” corresponding Boolean gate (last SS/CD45 plot, “blasts” circle zone, left or right Fig. 1A), conﬁrming there was no pathological cell in the sample.

In order to further evaluate the technical performances of the dry format tube, we mixedﬁve pathological bone marrow samples at diagnosis into non- pathological bone marrow containing immature B cells (hematogones) for detection quantitation of blast cells mixed with physiological B cell precursors (CD10 +/CD19+ characterized cells). We tested each diluted sample (1/10 and 1/100 dilution) with both standard and Duraclone approaches and conﬁrmed that there is an excellent agreement between the two methods for blast detection (e.g., 1/10 diluted blast cells = 4.1%

FIG. 1. (A) Control bone marrow sample stained with liquid format reagents (left) and dried format reagents (right): granulocytes in red, monocytes in green, mature T lymphocytes in blue, mature B lymphocytes in purple and physiological B cell precursors (hematogones) in turquoise blue. The“Cell” gate allows the exclusion of nonviable cells, fragment cells, and residual red blood cells. Events within the“Cell”gate are then analyzed according to the expression of CD19 in order to isolate B lymphoid cells. CD19 positive cells are studied through biparametric cytograms showing CD10 expression for immature B cells. The last cytogram (SS/CD45) shows events resulting from a Boolean gate, which includes regions as expected for typical blast cells in B-ALL based on diagnosis characterization (on CD10/CD38 and CD10/CD58 cytograms for example), and excludes mature B lymphocytes (on CD10/CD20 cytogram). (B) Quantitative correlation of the CD19+/CD45+ ratio; dried reagent tube on the X axis and liquid tube on the Y axis. Red dots represent PB and blue dots represent BM samples. (C) Bone Marrow MRD positive sample: stained with liquid format reagents (left) and dried format reagents (right). Blasts are represented in black, B mature lymphocytes in purple and hematogones in turquoise blue. MRD percentage was evaluated at 0.03% by the two approaches. (D) MRD quantitative concordance between the two approaches (dried or liquid). Red dots represent PB and blue dots represent BM samples.

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[dried tube] vs 4.2% [liquid tube]; 1/100 diluted blast cells = 0.3% [dried tube] vs 0.3% [liquid tube]) (Data not shown).

Comparison and validation of the RE-ALB tube performances

Twenty-nine cellular suspensions from B ALL patients were next analyzed: 10 derived from PB and 19 from BM aspirates. For these samples, we analyzed the number of CD45+ and CD19+ events, calculated a CD19+/CD45+ ratio, and compared this ratio between the two methods, using nonparametric Spearman correlation statistics. As expected, we found a strong correlation between liquid and dry reagents (r = 0,9747;

p < 0.0001) (Fig. 1B).

Then, we tracked potential blast cells in all samples tested in duplicate and concluded whether MRD was detected or not. The minimal amount of detectable blasts required to consider MRD as positive was a cluster of 10 to 20 events and the MRD result was given as the percentage of blasts present within total nucleated cells expressing CD45. If the MRD is undetectable, the detection threshold depends on the total number of analyzed cells. PB MRD assessment was performed at early time points of treatment (during or just after the prephase of corticotherapy) explaining the lower number of cells analyzed in PB samples. We were able to analyze at least 20,000 cells in the PB and at least 200,000 cells for the BM samples (in order to reach usually expected thresholds of 10⁻³ to 10⁻⁴, respectively).

To reach the highest possible detection performance, as well as endorse the use of additional liquid markers in dried tube, MRD detection for both methods was always made with the use of an additional patient- speciﬁc LAIP marker (identiﬁed at diagnosis), allowed by the use of an additional PE-coupled antibody for each tube. Comparison of the two formats demonstrated excellent agreement. Indeed, 23 samples were found positive by both methods. An example of BM positive samples for both liquid and dried format reagents is represented in Figure 1C (left: liquid, right:

dried). MRD detection by both approaches was 0.03%.

For the 23 positive samples, MRD percentages were highly concordant between the two methods with r = 0.9913 and aPvalue <0.0001 (Fig. 1D).

We also tested two CSF in the context of extramedul- lary relapse suspicion of B-ALL, but only with the Dura- clone tube as we did not have enough sample volume to test it in duplicate. Despite a low cell count (between 10,000 and 20,000 in the tested samples), this approach allowed accurate detection and quantitation of pathological cells in CSF (Data not Shown).

In addition, six samples were negative by both methods with a comparable detection threshold in all cases (four samples with a 10⁻⁴ sensitivity threshold and two with a 10⁻³sensitivity threshold).

DISCUSSION

Studies of MRD are becoming central to the clinical management of patients with acute leukemia. Flow cytometry provides fast and reliable results, but discrepancies may appear due to antibody cocktails variability and technical errors. We have evaluated here results obtained with a single tube containing dried antibody cocktail for B ALL MRD detection and quantitation.

The basic approach of ﬂow cytometry-based MRD analysis is to differentiate the residual leukemic blasts from normal B cell precursors (BCP) also known as hematogones, and other B lymphoid cells, based on the aberrant expression or deviation from the normal pat- tern of expression of a variety of antigens (9,10). The Duraclone dried tube contains seven different antibodies including the CD45/CD19/CD10 triad used for almost every B ALL MRD detection; they are considered as “backbone” markers. Other markers such as CD20, CD34, CD58, and CD38, are useful markers allowing lymphocytes, lymphoblastic and hematogones cells dis- crimination. Additional markers can be used for up to 10 color upgraded tube, allowing additional leukemia associated immunophenotype (LAIP) detection (CD73, CD86…) (7,11,12).

According to the manufacturer, 10⁶ cells should be suspended in 100 μl for each test. This is in line with recommendations for MRD analysis (13) and what is applied in our laboratory. However, high level of cellularity could not be reached in every case, especially for PB MRD detection during or after corticotherapy.

Despite these various cellularity conditions, correlations between the two methods showed highly comparable results.

To our knowledge, few studies have been published using the dried tube technology to screen hematological malignancies. In 2015, a Canadian study showed qualitative and quantitative equivalency between dried and liquid reagents for screening lymphoproliferative syndromes (14). In 2017, these results have been confirmed by other authors who used a 10 color tube containing 15 dried antibodies for screening lymphoproliferative malignancies (15). Finally, some authors tested the Duraclone technology for screening lymphoproliferative and myelodysplasic syndromes with a 10-color 14-antibody tube, proving this tube was useful in 60% of cases with significant time and cost savings (16). Another study aimed to compare dried tube immunophenotyping with liquid reagent method, assaying mean fluorescence intensities (MFI) for both methods (17) in the context of lymphocytosis or immune deficiency. This study revealed comparable results for MFI obtained with dried and liquid tubes.

Here, on a clinical approach, we further investigated if the endpoint results could be correlated as well, in the context of BCP-ALL MRD detection. Consistently, MRD detection obtained with the Duraclone technology was highly comparable with the liquid-antibody tube

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method. One interesting point is our demonstration that using additional LAIP markers to upgrade to an eight-color tube led to detection of low MRD level, with strong correlation when compared to liquid format tube. We tested several potential blast markers, such as lineage aberrant markers (CD33 or CD13), or some more recently described aberrant markers for BCP-ALL such as CD49f, CD66c, or CD123 ((11); (12)). These additional markers allowed quite low detection threshold, with comparable detection performance for dried versus liquid tube.

To conclude, we report here thefirst work aiming to study B ALL MRD detection byflow cytometry using a multicolor dried antibody single tube. We demonstrate the applicability of the Duraclone RE ALB tube in daily routine as we have shown a qualitative and quantitative correlation with the conventional approach (liquid reagent). This technology offers time saving (no cocktail preparation needed), elimination of pipetting errors, reduces potential waste of antibodies and promotes interlaboratory standardization. Performances may even be increased using 9 or 10 color tube when combining with other LAIPs when possible. This dried tube could be particularly appropriate for early MRD evaluation or in case of relapse suspicion (blood or CSF), and complies for standardization effort and workflow optimization.

ACKNOWLEDGMENTS

The authors thank Beckman Coulter Society for pro- viding the speciﬁc tube “Duraclone Rare Events ALB”. Preliminary data from this study was presented (poster and short oral presentation) at French Cytometry Asso- ciation meeting in Reims, France, November 7, 2017.

The authors declare no conﬂict interest.

AUTHOR CONTRIBUTIONS

LB and DB performed cellular assays. LB, DB, IA, and ML analyzed data. LB, DB, VN, and ML wrote the article.

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