Minimal Residual Disease In Chronic Lymphocytic Leukemia Research Articles

CD160 receptor in CLL: Current state and future avenues.

CD160 is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein expressed on cytotoxic natural killer (NK) cells and T-cell subsets. It plays a crucial role in the activation of NK-cell cytotoxicity and cytokine production. It also modulates the immune system and is involved in some pathologies, such as cancer. CD160 is abnormally expressed in B-cell chronic lymphocytic leukemia (CLL) but not expressed in normal B lymphocytes. Its expression in CLL enhances tumor cell proliferation and resistance to apoptosis. CD160 is also a potential prognostic marker for the detection of minimal residual disease (MRD) in CLL, which is important for theclinical management of CLL, the prevention of disease relapse, and the achievement of complete remission. In this review, we present an overview of CD160 and its involvement in the pathophysiology of CLL. We also discuss its use as a prognostic marker for the assessment of MRD in CLL.

Artificial Intelligence Enhances Diagnostic Flow Cytometry Workflow in the Detection of Minimal Residual Disease of Chronic Lymphocytic Leukemia.

Simple SummaryFlow cytometric immunophenotyping is critical in detecting minimal residual disease (MRD) in patients with chronic lymphocytic leukemia (CLL). However, flow cytometric analysis is complicated and time-consuming. Herein, we evaluated the performance of a deep neural network (DNN) in detecting CLL MRD and whether it could improve the diagnostic workflow in a clinical laboratory setting. Our findings demonstrated that a hybrid DNN approach had high accuracy in detecting CLL MRD; it standardized the gating strategy and dramatically reduced gating time, and it could be fully integrated into the existing clinical laboratory.Flow cytometric (FC) immunophenotyping is critical but time-consuming in diagnosing minimal residual disease (MRD). We evaluated whether human-in-the-loop artificial intelligence (AI) could improve the efficiency of clinical laboratories in detecting MRD in chronic lymphocytic leukemia (CLL). We developed deep neural networks (DNN) that were trained on a 10-color CLL MRD panel from treated CLL patients, including DNN trained on the full cohort of 202 patients (F-DNN) and DNN trained on 138 patients with low-event cases (MRD < 1000 events) (L-DNN). A hybrid DNN approach was utilized, with F-DNN and L-DNN applied sequentially to cases. “Ground truth” classification of CLL MRD was confirmed by expert analysis. The hybrid DNN approach demonstrated an overall accuracy of 97.1% (95% CI: 84.7–99.9%) in an independent cohort of 34 unknown samples. When CLL cells were reported as a percentage of total white blood cells, there was excellent correlation between the DNN and expert analysis [r > 0.999; Passing–Bablok slope = 0.997 (95% CI: 0.988–0.999) and intercept = 0.001 (95% CI: 0.000–0.001)]. Gating time was dramatically reduced to 12 s/case by DNN from 15 min/case by the manual process. The proposed DNN demonstrated high accuracy in CLL MRD detection and significantly improved workflow efficiency. Additional clinical validation is needed before it can be fully integrated into the existing clinical laboratory practice.

The Application of Minimal Residual Disease in Hemato-Oncology: A Review of Its Current Utility in Trials, Regulatory Decisions and Clinical Practice

The concept of minimal residual disease (MRD) has become common across multiple hematological malignancies. However, there is considerable variation in the application of this concept to research activities and clinical practice across different diseases. We sought to investigate the current status of MRD testing in research, its standing with regulators, and its application in clinical practice, and interpreted the differences identified in light of the differentiating characteristics of each disease. Seven malignancies were considered: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).The technology used to detect MRD, or its near equivalents of major or deep molecular response (MMR) in CML, is under constant development; current techniques are based on polymerase chain reaction for all indications considered (including next-generation sequencing) or flow cytometry for four indications (ALL, AML, CML, and MM), with a role for imaging also proposed in MM.A review of the published literature (via PubMed) for each of the diseases identified multi-study meta-analyses describing the prognostic significance of MRD in ALL, CML, and MM; evidence of this relationship was based on analysis of individual studies for the other four diseases.In the context of the prognostic significance of MRD status, MMR is well established as an outcome in clinical trials in CML and is emerging as a trial outcome measure in other indications. A review of registered active Phase 2 and 3 clinical trials (via clinicaltrials.gov) found that MRD (or an equivalent such as molecular response) was listed as an outcome measure in 21% of trials in ALL, 17% in CLL, and 12% in MRD; this was more common in Phase 3 trials (15/40, 16/51, and 14/73 trials, respectively). MRD was listed as an outcome measure in 6% of trials in AML, 2% in DLBCL, and 6% in FL. Furthermore, the European Medicines Agency formally accepted MRD as an intermediate endpoint in CLL (effective from 2016), while MMR was recommended as a primary endpoint in CML in 2013; consultation is ongoing regarding acceptance of MRD in MM. The US Food and Drug Administration has led a number of workshops on the use of MRD in four indications (MM, CLL, AML, and ALL).Aside from academic interest and clinical development programs, the use of MRD testing to guide treatment is recommended in US and European clinical guidelines for ALL, CML, and the acute promyelocytic variant of AML, and in US guidelines for CLL; US guidelines for MM recommend assessment of MRD but do not suggest that the result should inform the course of treatment. For other malignancies, prognostic implications and utility in trial settings are acknowledged, but MRD testing is not recommended in practice.The role of MRD in a range of hematological malignancies appears to be evolving rapidly. MMR in CML has been important in trials and in clinical practice for a number of years, but has recently been followed by MRD in CLL, while MRD testing in MM is on the horizon. MRD evaluation is now an integral part of both adult and pediatric treatment protocols. The biological basis of each disease (targets for detection, location of disease), interacting with the available technology, treatments, and treatment intent, may have contributed to different speeds of uptake. Given the growing body of research on detection technologies and its role in other malignancies, we expect MRD testing to become increasingly widespread and its role to shift from prognosis to a key factor in determining treatment pathways. DisclosuresNo relevant conflicts of interest to declare.

Highly Sensitive and Accurate Assessment of Minimal Residual Disease in Chronic Lymphocytic Leukemia Using the Novel CD160-ROR1 Assay.

Undetectable minimal residual disease (MRD) in Chronic Lymphocytic Leukemia (CLL) has a favorable prognostic outcome compared with MRD that can be detected. This study investigated a flow cytometric assay (CD160-ROR1FCA) targeting the tumor-specific antigens CD160 and receptor tyrosine kinase-like orphan receptor 1 (ROR1), along with CD2, CD5, CD19, CD45. CD160-ROR1FCA was compared with the originally published 8-colour European Research Initiative for CLL (ERIC) gold-standard assay for CLL MRD detection. CD160-ROR1FCA had a limit of detection of 0.001% and showed strong correlation with ERIC (R = 0.98, p < 0.01) with negligible differences in MRD detection (bias -0.3152 95%CI 5.586 to -6.216). Using CD160-ROR1FCA, increased expression of both CD160 and ROR1 was found in Monoclonal B cell Lymphocytosis (MBL) compared to low-level polyclonal B-cell expansions (p < 0.01). Patients in CR and with undetectable MRD had a longer EFS (not reached) than those in CR but with detectable MRD (756 days, p < 0.01) versus 113 days in patients with partial remission (p < 0.01). Patients with MRD levels of >0.01 to 0.1% had a longer EFS (2,333 days), versus levels between 0.1 to 1% (1,049 days). CD160-ROR1FCA is a novel assay for routine CLL MRD measurement and for MBL detection. MRD status assessed by CD160-ROR1FCA after CLL treatment correlated with EFS.

Dual antibody immunohistochemistry: an efficient and sensitive tool for the detection of residual disease in chronic lymphocytic leukemia

Highly effective treatments for chronic lymphocytic leukemia (CLL) have the potential to reduce significant tumor burden to single cells and therefore require sensitive tools to assess for minimal residual disease (MRD) in bone marrow (BM) biopsies. Flow cytometry (FC) is the current gold standard for detection of MRD, but requires a specialized facility, specific antibody panels, collecting hundreds of thousands-to-millions of cells, and personnel with expertise in the analysis and interpretation of FC MRD data, which may not be feasible in many small laboratories. Dual-antibody immunohistochemistry (DA-IHC) can identify abnormal populations better than morphology alone, but its correlation with FC assessment is not known. Our aims are to characterize the efficacy of DA-IHC in assessing BM samples post-treatment and to compare results with FC to evaluate for residual disease in CLL. We collected 2-year post-therapy data from 33 CLL patients on two treatment protocols, chemoimmunotherapy (CIT) and single agent ibrutinib (IB), as well as BMs from 10 healthy volunteers as morphologic controls. BM biopsy specimens were examined for the presence or absence of CLL based on morphologic evidence of lymphoid infiltration and aberrant co-expression of CD5 and PAX5 DA-IHC. FC using a standard CLL antibody panel was performed in parallel. All IB patients had residual disease detected by DA-IHC and FC, although five patients (22%) were morphologically negative by routine hematoxylin and eosin (H&E) stain. Those without overt morphologic evidence of disease showed DA-IHC-positive interstitial single cells and were positive for residual disease by FC. The assessments by DA-IHC and FC were significantly correlated (p = 0.004). Four patients (40%) treated with CIT were morphologically negative by H&E, and two of these had no detectable CLL by either DA-IHC or FC. The other two patients had low-level disease detected by both DA-IHC and FC. The MRD levels identified by DA-IHC were correlated with those by FC (p < 0.0001). We characterized the efficacy of DA-IHC in assessing CLL post-treatment and found assessments by DA-IHC and FC were statistically significant in both the IB- and CIT-treated patients. These findings suggest DA-IHC could accurately detect residual disease in cases lacking morphologic evidence by H&E alone. DA-IHC may be a useful tool in current practice as another sensitive and efficient method to assess for MRD in CLL.

Minimal Residual Disease in Chronic Lymphocytic Leukemia: A New Goal?

In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with TP53 deletion/mutation or unmutated IGHV genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.

Achieving Minimal Residual Disease Negative Status in CLL - a Good Point to Start Thinking about Life without Treatment: How to Check MRD Status?

Patients diagnosed with chronic lymphocytic leukemia (CLL) do not necessarily have to undergo intensive treatment. The implementation of the therapy depends on patient condition and the form of CLL which is determined using predictive and prognostic factors. Both indolent and aggressive forms of CLL should be monitored. To improve survival and the quality of life it is very important to know how the organism responds to the treatment. For this purpose, the estimation of the minimal residual disease (MRD) - the pool of persistent leukemia cells after or at the time of treatment was introduced. These cells resistant to treatment can lead to recurrence of the disease. There are several methods for MRD assessment, however in every case it is necessary to know the initial status of the tested alterations (chromosome aberrations, surface protein expression, etc.). It allows for assessment whether the treatment eliminated the leukemic cells. The most common method for MRD evaluation in CLL is flow cytometry. Intensive development of molecular biology techniques can contribute to implementation of other extremely sensitive and specific methods. This review presents the most recent state of the art concerning minimal residual disease in CLL, including methods that are used for MRD assessment.

Regulatory Informatics Reveals Minimal Residual Disease Trends in Hematologic Malignancies.

The application of regulatory informatics to identify trends in clinical trials is a new methodology with the potential to shape drug development programs. As an endpoint in hematologic malignancies, minimal residual disease (MRD) measures the depth of response to treatment, and has been given initial attention in draft regulatory guidelines and workshops by health authorities in the EU and US. In this paper, we analyze hematologic malignancy trials by searching for terms in Trialtrove, parsing the data sets with ActivePerl, and determining significance with the statistical package R. We identify trends in the use of MRD compared to other endpoints and in different indications over time from 2005 to 2014. Results show that 2 trends of endpoint and MRD use emerge over time and the use of MRD as a primary endpoint is growing. We also take a case-study approach to investigate the functional context of MRD in chronic lymphocytic leukemia and show that it is predominantly used to determine MRD-negativity and also in correlation with other endpoints. The application of informatics tools to regulatory science can evaluate the role and context of MRD as it evolves in the changing pharmaceutical landscape.

Ibrutinib Treatment of Relapsed CLL Following Allogeneic Transplantation: Sustained Disease Response and Promising Donor Immune Modulation

Abstract Background: Treatment of relapsed chronic lymphocytic leukemia (CLL) with ibrutinib results in high rates of progression-free and overall survival. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), is FDA-approved for use in relapsed CLL. Ibrutinib may also modulate donor T cell alloimmunity via inhibition of interleukin-2-inducible kinase (ITK). Here we report the effects of ibrutinib salvage therapy in 5 CLL patients who relapsed following allogeneic hematopoietic cell transplantation (allo-HCT). In addition to minimal residual disease (MRD) response measurements, we also assessed donor T cell chimerism and donor B cell immune reconstitution following ibrutinib therapy. Methods: Five patients with high-risk CLL relapsed 1-8.5 yrs following allo-HCT. Four patients had never achieved donor CD3 T cell chimerism >95% following reduced-intensity transplant. Ibrutinib 420mg daily was started 1 mo-2 yrs after clinical relapse. Four of the 5 patients remain on ibrutinib with treatment courses ranging from 3-17 mos. CLL MRD was measured by IgH high-throughput sequencing (HTS) using the ClonoSIGHTTM test (Sequenta, Inc.), which has the sensitivity to detect 1 CLL clone per million leukocytes. Lymph node (LN) size was assessed by CT scan and reported as the sum of the products of the LN diameters (SPD). Donor B cell reconstitution was determined by IgH HTS quantification of total IgH molecules and unique IgH clonotypes. Results: Lymphocytosis was observed in all 5 patients following initiation of ibrutinib treatment, consistent with previous reports. In 2 patients who received >1 yr of ibrutinib treatment, lymphocytosis peaked at 3 wks and 8 wks after initiation of treatment and slowly declined thereafter, fully resolving within 1 yr (Fig 1A). All 4 patients with pathologic lymphadenopathy prior to treatment experienced dramatic LN reduction (Fig 1B; 68% average reduction in LN size after 3 mos on ibrutinib). The longest duration of follow-up is reported for patient SPN 3975, who had a 17p deletion and received ibrutinib for 39 mos. Treatment was discontinued after CLL MRD became undetectable by ClonoSIGHT (Fig 1C). Evidence of donor T cell immune modulation included achievement of full donor CD3 chimerism after 1 yr and resolution of oral and skin chronic graft-versus-host disease (GVHD) after 6 mos. Although this patient has been off ibrutinib for >8 mos, full donor chimerism persists and CLL MRD remains undetectable (Fig 1C). Before ibrutinib treatment, donor B cells (excluding the patient’s CLL clone) accounted for 1% of PBMC (Fig 1D). Furthermore, recovering B cells have diverse, low frequency IgH clonotypes (Fig 1E). Together, our findings show rapid, sustained, and diverse immune reconstitution without CLL recurrence following ibrutinib discontinuation. Conclusions: Ibrutinib provides effective salvage therapy for CLL relapse following allo-HCT. Post-transplant CLL relapse is often extra-nodal and our experience shows ibrutinib is effective in clearing both nodal and extra-nodal disease. Here we present one patient who stopped therapy after achieving MRD negativity and maintains undetectable disease 8 mos following ibrutinib discontinuation. Ibrutinib treatment demonstrates promising donor immune modulation by promoting full donor chimerism and resolution of chronic GVHD. Our five patient experience supports using ibrutinib in relapsed CLL patients following allo-HCT. Clinical trials are needed to determine the best duration of ibrutinib therapy following post-allo-HCT relapse, chronic GVHD treatment efficacy, and the role of ibrutinib maintenance following allo-HCT. Download : Download high-res image (120KB) Download : Download full-size image Figure 1 . (A) Percent change in absolute lymphocytic count (ALC) for 2 patients who received ibrutinib treatment for >1 yr. SPN = Stanford Patient Number. (B) Percent reduction in LN size, as reported by the sum of the product of LN diameters (SPD) for 4 patients following initiation of ibrutinib. (C) CLL MRD (reported as a percentage of WBCs) and blood donor CD3 T cell levels shown for patient SPN 3975. (D) B cells (excluding the CLL clone) as percent of total PBMC for patient SPN 3975 as measured by IgH HTS. (E) Total IgH molecules and unique IgH clone counts for patient SPN 3975 at different time points (D=day) post allo-HCT. Disclosures Kong: Sequenta, Inc.: Employment, Equity Ownership. Klinger: Sequenta, Inc.: Employment, Equity Ownership. Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Coutre: Pharmacyclics, Inc.: Consultancy, Research Funding. Miklos: Sequenta, Inc.: Research Funding.

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10−4 to 10−5. One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups.

Ibrutinib Treatment of Relapsed CLL Following Allogeneic Transplantation: Sustained Disease Response and Promising Donor Immune Modulation

Background: Treatment of relapsed chronic lymphocytic leukemia (CLL) with ibrutinib results in high rates of progression-free and overall survival. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), is FDA-approved for use in relapsed CLL. Ibrutinib may also modulate donor T cell alloimmunity via inhibition of interleukin-2-inducible kinase (ITK). Here we report the effects of ibrutinib salvage therapy in 5 CLL patients who relapsed following allogeneic hematopoietic cell transplantation (allo-HCT). In addition to minimal residual disease (MRD) response measurements, we also assessed donor T cell chimerism and donor B cell immune reconstitution following ibrutinib therapy.Methods: Five patients with high-risk CLL relapsed 1-8.5 yrs following allo-HCT. Four patients had never achieved donor CD3 T cell chimerism >95% following reduced-intensity transplant. Ibrutinib 420mg daily was started 1 mo-2 yrs after clinical relapse. Four of the 5 patients remain on ibrutinib with treatment courses ranging from 3-17 mos. CLL MRD was measured by IgH high-throughput sequencing (HTS) using the ClonoSIGHTTM test (Sequenta, Inc.), which has the sensitivity to detect 1 CLL clone per million leukocytes. Lymph node (LN) size was assessed by CT scan and reported as the sum of the products of the LN diameters (SPD). Donor B cell reconstitution was determined by IgH HTS quantification of total IgH molecules and unique IgH clonotypes.Results: Lymphocytosis was observed in all 5 patients following initiation of ibrutinib treatment, consistent with previous reports. In 2 patients who received >1 yr of ibrutinib treatment, lymphocytosis peaked at 3 wks and 8 wks after initiation of treatment and slowly declined thereafter, fully resolving within 1 yr (Fig 1A). All 4 patients with pathologic lymphadenopathy prior to treatment experienced dramatic LN reduction (Fig 1B; 68% average reduction in LN size after 3 mos on ibrutinib). The longest duration of follow-up is reported for patient SPN 3975, who had a 17p deletion and received ibrutinib for 39 mos. Treatment was discontinued after CLL MRD became undetectable by ClonoSIGHT (Fig 1C). Evidence of donor T cell immune modulation included achievement of full donor CD3 chimerism after 1 yr and resolution of oral and skin chronic graft-versus-host disease (GVHD) after 6 mos. Although this patient has been off ibrutinib for >8 mos, full donor chimerism persists and CLL MRD remains undetectable (Fig 1C). Before ibrutinib treatment, donor B cells (excluding the patient’s CLL clone) accounted for <0.2% of total PBMC as determined by IgH HTS. Following discontinuation of ibrutinib, the percentage of donor B cells increased within 6 months, and now comprises >1% of PBMC (Fig 1D). Furthermore, recovering B cells have diverse, low frequency IgH clonotypes (Fig 1E). Together, our findings show rapid, sustained, and diverse immune reconstitution without CLL recurrence following ibrutinib discontinuation.Conclusions: Ibrutinib provides effective salvage therapy for CLL relapse following allo-HCT. Post-transplant CLL relapse is often extra-nodal and our experience shows ibrutinib is effective in clearing both nodal and extra-nodal disease. Here we present one patient who stopped therapy after achieving MRD negativity and maintains undetectable disease 8 mos following ibrutinib discontinuation. Ibrutinib treatment demonstrates promising donor immune modulation by promoting full donor chimerism and resolution of chronic GVHD. Our five patient experience supports using ibrutinib in relapsed CLL patients following allo-HCT. Clinical trials are needed to determine the best duration of ibrutinib therapy following post-allo-HCT relapse, chronic GVHD treatment efficacy, and the role of ibrutinib maintenance following allo-HCT. [Display omitted] DisclosuresKong:Sequenta, Inc.: Employment, Equity Ownership. Klinger:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Coutre:Pharmacyclics, Inc.: Consultancy, Research Funding. Miklos:Sequenta, Inc.: Research Funding.

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

IntroductionOver the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). ObjectiveTo compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. MethodsBetween October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. ResultsTo establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001).54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). ConclusionMonitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods.ReferencesFarren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183.Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures:Farren:BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

High-Throughput Immunoglobulin Gene Sequencing Quantifies Minimal Residual Disease in CLL with 10e-6 Sensitivity and Strongly Predicts Relapse After Allogeneic Hematopoietic Cell Transplantation

Abstract 2542 Background:Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR. Methods:We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient’s unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR. Results:CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days). Conclusions:Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment. Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.

Normal levels of peripheral CD19+CD5+ CLL‐like cells: Toward a defined threshold for CLL follow‐up—A GEIL‐GOELAMS study

The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

3.2 Evolution of High-Sensitivity, Multi-Color Flow Cytometric Immunophenotyping for Minimal Residual Disease Detection in Chronic Lymphocytic Leukemia: Peripheral Blood versus Bone Marrow?

Background: Detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) patients has become increasingly important as modern chemoimmunotherapy (CIT) regimens have become effective in reducing tumor burden or even eliminating detectable disease. From a clinical perspective, MRD status posttreatment has been shown to predict durability of remission. Current 6and 8-color flow cytometry assays can easily detect CLL cells to the 0.005% level (1 in 20,000 events). Historically, peripheral blood (PB) has been the standard source for evaluating CLL, while bone marrow (BM) studies have been used primarily to evaluate patients with unexplained cytopenias. A previous study based on 4-color flow cytometry analysis suggested a high concordance (87%) of MRD analysis between PB and BM as long as patients had not received monoclonal antibody therapy in the preceding 8-12 weeks. The goal of the present study was to compare MRD detection in PB and BM from CLL patients after first-line CIT using a highly sensitive 6-color flow assay to determine whether there were any differences in MRD levels. Methods: Thirty-one paired PB and BM specimens were obtained from 25 patients with a confirmed diagnosis of CLL. Twenty-one paired samples were obtained from 15 previously untreated patients on a phase II clinical trial evaluating lenalidomidebased consolidation therapy after 6 cycles of pentostatin, cyclophosphamide, and rituximab induction therapy. Ten paired samples were from previously untreated patients on a phase II clinical trial evaluating pentostatin, cyclophosphamide, and ofatumumab. Twentyeight paired specimens were obtained 3 months after rituximab/ ofatumumab and the remaining 3 specimens were obtained 8-12 weeks following rituximab therapy. Individual patient samples were processed and analyzed on the same day. High-sensitivity flow cytometric immunophenotyping was performed in all specimens. A total of 500,000 events were collected in all cases and analyzed on a Becton-Dickinson Canto II flow cytometer. The standard single-tube, 6-color antibody panel included CD45, CD19, CD20, CD5, and kappa and lambda immunoglobulin light chains; additional antibodies were added as needed. Positive events were based on the identification of lymphoid cells by light scatter followed by gating on CD19positive B-cells, then dual expression of CD20(lo)/CD5 cells, and finally evaluation for kappa/lambda monoclonality. The level of MRD was assessed by percentage MRD calculated as (CLL events)/ (total live, non-aggregated WBCs) 100. This assay was developed in a clinical laboratory and validated by comparing it to a previous standard 4-color assay in 562 specimens (unpublished data). Validation studies confirmed a higher level of sensitivity with the 6-color assay and the ability to consistently detect MRD or monoclonal B-cell lymphocytosis events to the 0.005% level (25/500,000 events). Results: PB and BM MRD analysis was concordant in 20/31 (65%) paired samples using the single-tube, 6-color antibody analysis; 15/31 paired samples had detectable MRD in both PB and BM, and 5 had no detectable MRD in either PB or BM. Eleven of 31 (35%) studies showed detectable MRD in BM, but no CLL cells in PB. Considering BM MRD analysis as the standard, the sensitivity of the PB MRD assay in this cohort was 58% (15/26). The amount of MRD was on average 5-fold greater in BM as compared to PB. The level of MRD in BM (range 0.006-6.008%; median 0.765%) was greater than in PB (range 0.035-38.726%; median 0.161%) in all cases, with a median difference of 0.343% (p 0.0054). In the 15 paired specimens with MRD in both PB and BM, the level of residual disease was 1% in 12/15 PB and 10/15 BM specimens. Among the 11 paired samples in which MRD was only detected in BM, 10 (91%) had 1% clonal B-cells present on the BM MRD assay, with positivity ranging from 0.005% to 3.231% (median 0.061%); this was 10 times lower than the median value of BM MRD for samples in which both PB and BM were MRD-positive, indicating a lower level of disease burden in the group with discordant MRD results on BM and PB analysis. Conclusion: A single-tube, 6-color flow assay is a highly sensitive approach for the detection of MRD in CLL. The level of MRD following effective CIT was 5-fold higher in BM as compared to PB. A negative PB MRD study cannot be used as a surrogate for the absence of BM disease in CLL patients.

High-Throughput VDJ Sequencing Is Superior to Quantitative PCR and Flow Cytometry for the Quantification of Minimal Residual Disease In Chronic Lymphocytic Leukemia After Hematopoietic Cell Transplantation.

AbstractAbstract 1290 Background:The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention such as chemotherapy or donor lymphocyte infusion to prevent clinical relapse. Validated methods for MRD assessment include allele-specific oligonucleotide quantitative PCR (ASO Q-PCR) and flow cytometry (FC). ASO Q-PCR affords high-sensitivity, but is laborious, time-intensive, and not widely available due to its dependence on patient-specific primers. FC is widely available, but is expensive and has lower sensitivity. Boyd et al. (Sci Transl Med 2009;1:12ra23) demonstrated the feasibility of high-throughput sequencing (HTS) of the immunoglobulin heavy chain variable region (IgVH) VDJ segment for quantifying CLL MRD using ASO Q-PCR as a reference standard. In the current study, we compare HTS, ASO Q-PCR, and FC for tracking a group of CLL patients at a series of time points following HCT. Methods:We performed HTS using BIOMED-2 (Van Dongen et al., Leukemia 2003;17:2257) framework 1 (FR1) and 2 (FR2) consensus VH primers with a consensus JH primer. PCR products were sequenced on a 454 pyrosequencer. We tested the sensitivity of both primer sets by creating serial dilutions of a FACS-sorted CLL population into a donor leukapheresis product with known B cell composition such that the CLL cells were as low as 10e-5cells. For our comparisons of HTS, ASO Q-PCR, and FC, we tested a total of 29 samples from 6 CLL patients at up to 10 different time points following HCT. Results:Although all 6 patients were found to have unmutated IgVH using conventional analysis, we discovered single amino acid sequence variants in up to 17% of CLL-related sequences using HTS. We verified that these changes were not the result of technical artifact by detecting the same oligoclonal species at several time points. For our analyses, we thus defined the “CLL clone” as the dominant sequence plus those sequences of the same CDR3 length that differed by up to one amino acid. In a normal donor, HTS with FR1 and FR2 primers respectively amplified 93.7% and 91.7% of expected V-J combinations (R2 = 0.92), indicating broad repertoire coverage. When purified CLL cells were serially diluted to 10e-5 in a normal donor population, we detected the CLL clone with both primer sets when we sequenced the IgVH VDJ with 150,000 reads, confirming 10e-5 sensitivity. ASO Q-PCR demonstrated the same level of sensitivity in this dilution series. In order to compare FC to the DNA-based techniques, we transformed FC data to represent it as CLL cells per microgram of total genomic DNA per cell. FC under-represented MRD levels by up to 80% in comparison to HTS and ASO Q-PCR. The PCR-based techniques were in accord when residual disease was low (R2 = 0.88). At high disease burdens, the dynamic range of HTS quantification is limited by the number of sequence reads dedicated to those specimens; however, HTS data permits analysis of CLL clones per total VDJ sequences which appears to be useful for clinical interpretation in such scenarios. Finally, HTS revealed VDJ repertoire reconstitution and hypermutation kinetics. In 3 patients who received post-HCT Rituximab (RTX) for GVHD prophylaxis, 6.0 +/− 0.6% of sequences were hypermutated at day +365 versus 17.8 +/− 3.0% in those who did not receive RTX (p = 0.005), confirming expected delays in immune reconstitution following post-HCT RTX therapy. Conclusions:HTS exhibits 10e-5 sensitivity for CLL MRD assessment, which is equivalent to ASO Q-PCR and superior to FC. Further validation using material from 35 CLL patients previously evaluated by ASO Q-PCR is underway. We anticipate HTS will broaden the availability of MRD assessment by providing high sensitivity using generally applicable consensus primers. HTS also has the benefits of detecting somatic mutations and revealing rich data regarding VDJ repertoire reconstitution. Furthermore, HTS of B and T cell immunoreceptor genes using consensus primer sets has the potential to provide MRD assessment of all lymphoid malignancies following HCT. Disclosures:No relevant conflicts of interest to declare.

Inter-Laboratory Assessment of Minimal Residual Disease In Chronic Lymphocytic Leukemia – Evaluation of the Novel CD160FCA Assay

AbstractAbstract 2723With the advent of highly effective chemo-immunotherapy regimens, the treatment of chronic lymphocytic leukemia (CLL) is no longer simple palliation but aims to achieve a minimal residual disease (MRD)-negative remission. Historically, CD5 and CD19 with demonstration of clonality was the mainstay of MRD evaluation in CLL. However, this approach is limited by the presence of normal B1-cells (CD5+CD19+) and the inability to demonstrate light-chain restriction with very low B-cell numbers. More recently, an international standardized approach (ISA) to the assessment of MRD in CLL was published with a detection accuracy of 95.7% for the detection CLL above 0.01%, involving 8 monoclonal antibodies (mAbs) in a 5 test system.1 CD160 is an activatory NK cell receptor, but is not expressed on normal B-lymphocytes, B1-cells, naive B-cells or activated B-lymphocytes. CD160 is expressed on malignant B-cells in >98% of cases of CLL, but in only 15% of non-CLL cases. This makes CD160 a unique marker for the assessment of MRD. Methods:Utilizing the anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive CD160 Flow Cytometric Assay (CD160FCA) incorporating CD2, CD5, CD19, CD23 and CD160. Whole blood (1×106 leukocytes) were labeled with mAbs, followed by an ammonium chloride-based lysing method. Data acquisition was captured using a sequential gating strategy focusing only on the malignant population. The CD160FCA was evaluated in two centers and the results compared with the ISA methodology in two different centers. Results:As expected, light-chain restriction could not be reliably measured in patients with very low B-cell numbers. 23 cases were analyzed at four centers. Concordant results were found in 22/23 cases with a sensitivity of 1.00 (P=0.0023, OR 91.00). Of the 23 patients 87% were MRD positive by the CD160FCA compared to 82% by the ISA. There were no cases that were MRD positive by the ISA and negative by the CD160FCA. There was a close correlation between the two methodologies for disease levels above 0.01% (Spearman Rank R=0.9913, P<0.0001). When studying the median fluorescence intensity (MFI) of CD160, the MRD positive cases had a mean MFI of 374 (288-461 95%CI) and the negative cases 47 (2-93 95%CI) (Figure 2). The CD160 FCA offers a single tube analysis for MRD, which is highly sensitive, independent of the therapy and can be employed throughout treatment. Importantly, not only is this approach simpler, cheaper and faster than current MRD approaches, it is still informative in cases where no light chain clonal population is found, or where restriction studies have failed. With increasing evidence that MRD detection in CLL can predict patient outcome, CD160FCA represents a simple tool for MRD assessment. Reference:Rawstron AC, Villamor N, Ritgen M et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 2007;21(5):956-964. [Display omitted] [Display omitted] Disclosure:No relevant conflicts of interest to declare.

The Immunophenotype Signature CD49d+CD38+ Identifies Chronic Lymphocytic Leukemia Cases with a Higher Potential for Migration Beneath Marrow Stromal Cells.

Abstract 356▪▪This icon denotes an abstract that is clinically relevant.Adhesion of chronic lymphocytic leukemia (CLL) cells to stromal cells in the marrow and secondary lymphoid tissues confers drug resistance and may account for survival and maintenance of residual CLL cells after conventional treatments, paving the way to relapses. Therefore, targeting the cross talk between CLL cells and stromal cells represents an attractive and necessary approach to overcome minimal residual disease in CLL. We previously reported that fractions of CLL cells and other neoplastic B cell spontaneously migrate beneath marrow stromal cells (MSC) in a CXCR4- and CD49d-(VLA-4) dependent fashion, an in vitro phenomenon termed pseudoemperipolesis (PEP). Also, we reported that cells that migrated beneath and underneath MSC were largely protected from cytotoxic drugs, in contrast to cells that remained in the supernatant (Blood 113:4604-13, 2009). The aim of this study was to identify correlations between CLL surface markers and their ability to migrate beneath MSC.We tested samples from 116 different CLL patients in co-culture assays for their ability to migrate beneath MSC (KUSA H1 cells). After 6 hours of incubation, CLL cells were vigorously washed off the MSC layers, and PEP+ cases were identified by phase contrast microscopy as cases, in which CLL cells were visualized within the stromal layers, characterized by the dark appearance of CLL cells that had migrated into the same focal plane as the MSC. We characterized 45 PEP+ and 71 PEP- CLL cases. Next, we profiled the expression of adhesion molecules and chemokine receptors, including CD44, CD49d, CD62L, CXCR3, CXCR4 and CXCR5 by flow cytometry in the PEP+ and PEP- subgroups. Based on the mean fluorescence intensity ratios (MFIR), we found that both groups displayed comparable levels of CD44 (58.8±4.6 in PEP+ vs. 58.7±3.5 in PEP-), CD62L (7.8±1.3 vs. 9.3±1.3), CXCR3 (7.9±1.0 vs. 8.0±0.8), CXCR4 (87.6±9.3 vs. 81.4±4.6), and CXCR5 (47.4±2.9 vs. 49.3±2.7). In contrast, the levels of CD49d were significantly different between these two groups; the MFIR for CD49d was 14.0±2.4 in PEP+ cases and 4.0±0.7 in PEP- cases (p<0.0001, see Fig. 1). Interestingly, in PEP+ cases, higher levels of CD49d correlated with lower levels of CXCR4 (r=-0.43; p<0.003), which could be due to higher affinity to a CXCL12-rich microenvironment, with respective CXCR4 down regulation. Analysis of prognostic factors (CD38, ZAP-70 and mutational status) revealed that PEP+ cases displayed higher level of CD38 expression: 29.8±5.8% vs. 15.6±2.7% (MFIR, p<0.05), whereas there was no difference in ZAP-70 expression or mutational status between PEP+ or PEP- cases. When comparing cytogenetic profiles, favorable risk (del13q14.3 and normal karyotype) cases represented 43.6% in the PEP+ vs. 71.2% in PEP- groups; trisomy12 cases 23.1% vs. 4.5%, and poor risk cytogenetics (del11q, del17p, complex) represented 33.3% in the PEP+ vs. 24.3% in PEP- group.Collectively, the distinct immunophenotype of CD49d+CD38+ identifies CLL cases with a higher affinity for migration beneath MSC. Consequently, CLL cells in such cases are expected to be more difficult to eradicate by conventional treatments because of higher levels of stroma-mediated drug resistance. Drugs, such as Natalizumab or Plerixafor, that target CLL-stroma interactions, could be of particular benefit for such patients by disrupting CLL-stroma crosstalk and mobilization of CLL cells from protective marrow niches. Disclosures:No relevant conflicts of interest to declare.

Detection of Minimal Residual Disease in Chronic Lymphocytic Leukemia with Monoclonal Antibodies Specific for CD5, CD10, CD19, and ROR1.

ROR1 is an oncofetal protein expressed in chronic lymphocytic leukemia (CLL). We generated a monoclonal antibody (mAb) specific for ROR1 (4A5) and used this to stain blood or marrow cells from patients with CLL and other adults. We found that 4A5 reacted with the CLL cells of all patients examined and also with some cells that had an immunophenotype of B lymphocyte precursors (BLP) (CD10+, CD19+, CD20-variable, dim CD45+, surface immunoglobulin-negative). 4A5 showed no reactivity with other blood or marrow cells. We speculated that this mAb could be used to detect CLL cells in blood or marrow of patients after therapy, providing a means with which to assess minimal residual disease (MRD). For this purpose, we evaluated a four-color combination of mAbs using CD10-FITC, CD19-PE, CD5-PerCP-Cy5.5, and 4A5-Alexa-647. MRD detection limits were established through reconstitution studies in which we made serial dilutions of CLL cells into normal blood or marrow samples. MRD was measured by whole blood lysis and acquisition of 100,000 events using a FACSCalibur and a gating strategy designed to exclude CD5-negative, CD10-positive, and 4A5-negative B lymphocytes (CD19+). CLL cells stained with 4A5 had a mean fluorescence intensity (MFI) of 31 (N=100) and a MFI ratio (MFIR) relative to that of isotype control stained CLL cells of more than 10:1. On average, 94% of the CLL cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 88–98%). Marrow samples (N=9) from adults with diagnoses other than CLL and that had 1–10% BLP had CD5− negative, CD10+, CD19+ cells that reacted with the 4A5-Alexa 647. These cells in such samples had a MFI of 19 and a MFIR relative to that of isotype-control-stained BLP of 6.3. On average, 48% of such cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 18–79%). Four color flow cytometric analysis with CD10, CD19, CD5, and 4A5 detected CLL cells present at less than 0.1% in reconstituted blood or marrow samples, including marrow with 3–5% BLP. Background was between 0.01% and 0.1% with whole blood (marrow) lysis when we acquired a total of 100,000 flow-cytometric events. These data indicate that anti-ROR1 mAbs can be used with mAbs specific for CD10, CD19, and CD5 for sensitive detection of MRD in CLL.