A comparison of flow cytometry detection of minimal residual disease and chimerism kinetics in chronic lymphocytic leukemia patients after allogeneic hematopoietic stem cell transplantation

Determination of minimal residual disease (MRD) remains crucial for the follow-up after therapy in chronic lymphocytic leukemia (CLL) patients. Chimerism was assessed by short tandem repeat (STR)PCR and single nucleotide polymorphisms (SNP)PCR, and MRD by a multicolor flow cytometric approach in 12 consecutive patients with CLL after they received allogeneic stem cell transplantation (SCT). Overall, 11 patients achieved MRD flow negativity [10 had full donor chimerism (FDC) and one had mixed chimerism (MC)]. Only one patient remained with MRD flow positivity and displayed MC. Fiftysix samples were concomitantly studied by both chimerism and MRD flow. A significant correlation was observed between MRD flow data and chimerism in both PB and BM by using a mixed effect linear regression (p < 0.001). Flow cytometry approach of MRD can be easily combined with chimerism during the follow-up post-allogeneic SCT. Both techniques appeared complementary for guiding post-transplant immunomodulation.


INTRODUCTION
Important advances have been made over the past two decades in the prognosis and treatment of chronic lymphocytic leukemia (CLL) [1].New therapeutic approaches aim to induce molecular remission.The better quality of response, provided by novel agents and new therapeutic approaches, requires necessarily more sensitive tools for precise remission assessment [2][3][4][5][6][7].The evaluation of response to treatment in CLL is currently based on the National Cancer Institute (NCI) criteria [8].Several studies have demonstrated that CLL patients achieving response at a molecular level without any detectable minimal residual disease (MRD) have a longer survival [5][6][7][8][9].Using a very high sensitive flow cytometry technique, several groups have shown that patients in complete remission (CR) with detectable MRD have an increased risk of early relapse [3,6].In this setting, bone marrow biopsy appeared to be less sensitive than flow cytometry [10].Conversely, similar results have been shown with real-time quantitative (RTQ) polymerase chain reaction (PCR) and MRD flow monitoring in terms of leukemia cell clearance kinetics and timing for reaching MRD negativity [6].
Because of the relatively advanced age of patients with CLL, no consistent efforts have been made to conduct stem cell transplantation (SCT) with use of allogeneic donors.However, allogeneic SCT remains the only curative treatment for CLL in younger adults.In the setting of allogeneic SCT, molecular techniques for chimerism determination are routinely used for engraftment follow-up and early prediction of post-transplant relapse.They have been routinely used in parallel with MRD analysis by flow cytometry.Furthermore, allogeneic SCT after reduced intensity conditioning (RIC) has been recently developed as a safer approach for older patients and has therefore emerged as the treatment of choice for older high-risk CLL patients requiring transplantation [11].In this setting, the significance of chimerism and MRD flow cytometry needs to be reevaluated.The aim of the present study was to compare chimerism and MRD flow cytometry level after allogeneic SCT in order to fully document CR and to guide immunomodulation by donor lymphocyte infusions (DLI).

Patient Characteristics
Between 2000 and 2007, 12 CLL patients (9 males, 3 females) with a median age of 51 years (range 31 -66 years) underwent allogeneic SCT in our institution.At the time of diagnosis, 2 patients were in stage A, 6 in stage B and 4 in stage C according to Binet's classification [12].Matutes' score was noted at 5 for 7 patients, 4 for 2 patients and 3 for the last 3 patients.Before allogeneic SCT, one patient received one line of conventional therapy and 11 received more than 2 lines: 11 patients received fludarabine-based therapeutic regimens, 6 received immunotherapy with rituximab and/or alemtuzumab, 3 have previously been auto-transplanted, and one has received a first allogeneic SCT.At the time of allogeneic transplant, 3 patients were in CR, while 7 were in partial remission (PR) and 2 in progressive disease.Response criteria were defined according to NCI guidelines for CLL [8].

Graft-versus-Host Disease
After transplant, 6 patients developed a grade 2 (n = 3) or 3 (n = 3) acute GVHD and 8 patients developed a chronic GVHD (4 limited and 4 extensive).The patient allografted twice presented after the second SCT, a grade 2 acute GVHD followed by a limited chronic GVHD.At the time of the last follow-up [median, 53 months (range: 3 -82 months)], 2 patients had died from infections, while 10 were still alive.

Chimerism Analyses
Chimerism was documented on days 30, 60, 90, and on months 6 and 12 during the first year following SCT, and then twice a year.Chimerism was assessed on total PB cells, total BM cells, selected CD3 The RT quantitative PCR chimerism assay, using the Taqman technology and ABI 7700 (Applera), has been previously described [14].Here, mixed chimerism was defined by the presence of at least 0.1% of RC.

MRD Flow Cytometry Analysis
MRD flow cytometry was assessed, every 3 months during the first year following transplant, and every 6 months afterwards, on PB and/or BM cells by an international standardized multicolor approach (S: 0.01%) [7].
Overall, 65 PB MRD flows, and 23 BM MRD flows were performed with a median of 5 PB MRD analyses (range: 1 -20), and 2 BM MRD analyses (range: 1 -5) per patient.Patients were considered MRD flow positive if the detected MRD CLL levels were ≥ 10 -4 .Specificity and sensibility of the technique were validated by using a series of 10 normal PB and BM samples.MRD was studied by using quadruple antigenic combinations: CD20-FITC/ CD79b-PE/ CD19-PerCP-Cy5.5/CD5-APC, CD43-FITC/ CD79b-PE/ CD19-PerCP-Cy5.5 CD5-APC, Kappa-FITC/Lambda-PE/CD19-PerCP-Cy5.5/CD5-APC, and CD22-FITC/ CD5-PE/ CD19-PerCP-Cy5.5/CD38 APC.In BM samples, the combination containing CD38 was used to better discriminate CLL cells from B-cell precursors (hematogones).In addition to daily calibrations using Calibrite beads (Becton Dickinson) and FAC-SComp v4.2 software (Becton Dickinson), isotype controls were used to optimize light scatter, amplification and threshold.For our study, at least 50 CLL cells forming a population in light scatter in at least two of the three MRD tests (excluding K/L antigens) were required as evidence for MRD (absolute specificity threshold).The result was classified as positive if the level was above the mean number of the false positive cells in healthy controls.A median of 300,000 leucocytes/samples (range 50,000 and 600,000) depending on time point was acquired using CellQuest Pro software (Becton Dickinson) and analyzed using Paint-A-Gate software (Becton Dickinson).The complete gating strategy for identification of CLL specific phenotype (MRD flow assay) has been previously described, using a CD43 + as fourth criterion for CLL cells [5].

Statistical Analysis
Kaplan Meier product-limit estimates were used to assess the probability of overall survival (OS).The relationship between chimerism and MRD flow assay was evaluated by a linear mixed effects model [15].The model included MRD as the resulting variable, chimerism as the fixed effect and patients as the random effect.An analysis was performed for BM and a second analysis was performed for PB.Significative effects were defined by p values of less than 0.05.All analyses were performed using Splus 6.2 (Insightful, Seattle).

RESULTS
At the time of study, the median follow-up was 53 months.Two patients had died from infection, while 10 were still alive.The 2 patients who died displayed MDR negativity and had FDC.Among alive patients, 9 were with MRD flow negativity (8 had FDC and one had MC), while one was with MRD flow positivity and displayed MC.Two hundred and fifty-one samples were tested for chimerism (192 from PB and 59 from BM) and 88 for MRD flow cytometry (65 from PB and 23 from BM).Only 56 samples were studied at the same time point, and were then used for comparing quantitative MRD flow cytometry and chimerism.

Chimerism Analysis
Comparison of results obtained from PB and BM samples showed 93% of concordance.The conversion of mixed chimerism (MC) to full donor chimerism (FDC) at Day 30 post-transplant was observed in 5 cases.The conversion was observed between Day 30 and Day 120 in 3 cases and after Day 120 in one case.Three patients never converted to FDC (patient # 12 had not enough follow-up, patient # 9 remained in stable MC at 3 months post-transplant, and patient # 8, who relapsed after a first RIC SCT, underwent then a second allogeneic SCT from another HLA identical sister allowing a durable FDC).Eleven patients had their chimerism done on selected cells, of whom 4 with MC (Table 2).

MRD Flow Cytometry Analysis
Comparison of results obtained from PB and BM samples showed 100% of concordance.Because of the recent introduction of MRD flow assay in the clinical routine, longitudinal data during the first year post-transplant were only available for 8 patients.They showed MRD flow negativity at Day 30 in one case, at Day 90 in another case, at Day 120 in 3 cases, and at 12 months post-transplant in 2 cases.One patient remained positive, but displayed decreasing MRD flow levels.Regarding the 4 patients without optimal longitudinal follow-up, all patients achieved MRD negativity but were only assessed at time points for which samples were available (on months 30, 36, 42, and 48 post-transplant, respecttively).Patient # 8 never achieved MRD negativity after the first transplant, but showed a low MRD level at 12 months and MRD negativity 24 months after the second SCT.

Comparison of Chimerism and MRD Kinetics
Results are summarized in Table 2. Regarding kinetics

DISCUSSION
Important advances have been made in the prognosis and treatment of CLL over the past two decades [1].With conventional therapy, a large proportion of patients can achieve CR [16,17].However, allogeneic SCT remains the only curative therapy [18,19].The number of allogeneic SCT performed in this disease is therefore susceptible to significantly increase, mainly due to the recent development of RIC allogeneic SCT in older patients [20,21].New technologies such as 4-color flow cytometry and RT quantitative PCR have identified the persistance of a detectable disease in patients considered in CR according to international guidelines [5].The absence of detectable leukemia by these sensitive tests seems to be an important prognostic factor [9,22] , since it has been shown correlated with a longer survival.A strong concordance has been described between these two techniques for MRD evaluation, with a limit of detection of one malignant cell per 10.000 total cells and 100% applicability of MRD flow assay.The present series, summarizing our experience in the setting of allogeneic SCT in CLL, tends to confirm previous reports.All patients, except one, showed MRD negativity when tested by flow cytometry.Ten patients obtained FDC, while 2 only achieved MC.We first established a positive concordance between PB and BM for both chimerism and MRD flow.Secondly, a positive concordance (89%) was confirmed between determination of residual disease by both flow cytometry and chimerism.Two types of conversion after transplantation were described for both chimerism and MRD flow: an earlier conversion following transplant and a later conversion.The kinetics of conversion to FDC was faster than kinetics of disappearance of detectable disease as observed by using flow cytometry: 42% of documented cases converted to FDC at Day 30, while at the same time only 12% of assessable cases were MRD negative by flow cytometry.Flow cytometry detection of MRD seems therefore more sensitive.Interestingly, detection of MRD by flow cytometry seemed also more acurate than total cell chimerism evaluation among the non concordant samples coming from one single patient.However, when considering lineage specific chimerism by selecting CD19 + cells for instance, a full donor profile quite concordant with MRD flow negativity was observed.
However, as no relapses were reported in the followup of our series, after reaching negativity, it is difficult to judge the relevance of the suggested use of MRD instead of chimerism in the long-term follow-up.The two techniques must certainly be considered as complementary for documenting MRD in CLL patients after allogeneic SCT and guiding immunomodulation.
The quantitative techniques used for the assessment of MRD and chimerism seemed therefore interesting tools for documenting disease and transplant status in CLL, as it has been also previously demonstrated in other hematological malignancies [23].
In the transplant setting for CLL patients, MRD detection should be considered as a surrogate marker for disease eradication.Our results are in agreement with previously published data showing the importance of MRD eradication assessed by flow cytometry during the posttransplant follow-up in CLL [2,3,6,10].However we reported only on a small series heterogeneous in terms of conditioning regimens, and chimerism results could be different after myeloablative and non-myeloablative conditioning.MRD flow appeared more specific than total cell chimerism evaluation.MRD flow assay also appeared as a faster and less expensive technique for guiding adoptive immunotherapy or treatment abstention after allogeneic transplantation.Nevertheless, both techniques appeared complementary, most specially when studying lineage specific documentation.Another important point is to consider a strict schedule of evaluation.

Table 1 .
Transplant characteristics of the 12 CLL patients.