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Published Ahead of Print on November 30, 2009, as doi:10.3324/haematol.2009.018911. Copyright 2009 Ferrata Storti Foundation. Early Release Paper Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease by Esther Domingo, Cristina Moreno, Alfonso Sánchez-ibarrola, Carlos Panizo, José Antonio Páramo, and Juana Merino Haematologica 2009 [Epub ahead of print] doi:10.3324/haematol.2009.018911 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Haematologica (pissn: 0390-6078, eissn: 1592-8721, NLM ID: 0417435, www.haematologica.org) publishes peer-reviewed papers across all areas of experimental and clinical hematology. The journal is owned by the Ferrata Storti Foundation, a non-profit organization, and serves the scientific community with strict adherence to the principles of open access publishing (www.doaj.org). In addition, the journal makes every paper published immediately available in PubMed Central (PMC), the US National Institutes of Health (NIH) free digital archive of biomedical and life sciences journal literature. Haematologica is the official organ of the European Hematology Association (www.ehaweb.org). Support Haematologica and Open Access Publishing by becoming a member of the European Hematology Association (EHA) and enjoying the benefits of this membership, which include free participation in the online CME program Official Organ of the European Hematology Association Published by the Ferrata Storti Foundation, Pavia, Italy www.haematologica.org

Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease In a recent paper by Béné and Kaeda, 1 technical approaches for minimal residual disease (MRD) assessment are extensively reviewed. PCR-based studies have proved to be 1-log more sensitive than flow cytometry (FC). For this reason, they are increasingly being considered preferable for MRD analysis, especially at the end of therapy or posthematopoietic stem cell transplantation. 1,2 It would be valuable to develop MRD flow cytometry assays with this level of sensitivity that could be applied routinely. In the present work, we analysed MRD + samples with a level of infiltration below the limit of detection of routine FC, which is accepted as 10-4 and comes from the standard acquisition of 2!5 " 10 5 leukocytes. 3-11 At least 10 fold more leukocytes must be acquired to increase sensitivity by one log; this large number of leukocytes can be acquired easily in digital cytometers by acquiring several individual tubes stained with the same combination of monoclonal antibodies, and appending them in a single file. Because the time of acquisition for each individual tube is not increased, no problems of cellular aggregation arise. Using this approach, we acquired 6 million leukocytes from each sample using a FACSCanto flow cytometer (Becton-Dickinson). The stability of fluorescent parameters between tubes was verified (Online Supplementary Figure S1). The study was performed with the event rate used routinely in our laboratory, i.e. 2000 events/sec. To explore the possibility of decreasing acquisition time, we analyzed the influence of increasing the event rate on the percentage of electronic aborts (due mainly to coincidence events), and compared MRD measurements in samples acquired at different event rates (Online Supplementary Table S1). We found that the acquisition event rate could be increased reliably to at least 4000 events/sec. Using this event rate, the time of acquisition of 6 million leukocytes is suitable for routine measurements. 1

We assessed that this approach enhanced sensitivity by 1 log by comparing MRD analysis by FC with real-time quantitative PCR for BCR-ABL1 p190 transcripts in serial dilutions of a Philadelphia cromosome-positive B-II acute lymphoblastic leukemia sample (Table 1A). Thereafter we selected multiple myeloma (MM) and B-cell chronic lymphocytic leukemia (B- CLL) MRD+ samples with about 0.01% infiltration and diluted them 10-fold with normal leukocytes. As shown in Table 1B, MRD was detected accurately in all diluted samples (10-5 infiltration). Observed values were within the expected ±10% in all cases. This increase in sensitivity did not compromise the specificity of the technique. Ten million leukocytes from the bone marrow of patients (n = 3) without hematological neoplasias and from the peripheral blood of healthy donors (n = 3) were acquired and blindly tested for the presence of events with a myelomatous or B-CLL phenotype, respectively. The results were unambiguously negative (Online Supplementary Figure S2). We also acquired 10 million normal leukocytes and searched for events with phenotypic characteristics of other hematological malignancies, such as follicular lymphoma and hairy cell leukemia, with negative results. In addition to the total number of leukocytes analysed, the sensitivity of FC depends on the number of neoplastic events that must be detected in order to obtain precise measurements. Based on the theory that rare events follow a Poisson distribution, it is accepted that 50-100 events are needed to reliably measure the frequency of a population. 3,7,8,11,12 However, to our knowledge, no studies have really measured the influence of the number of detected tumoral cells on MRD measurement precision. Accordingly, we determined the coefficient of variation (CV) of the percentage of MRD in samples from patients with MM, B-CLL and T- cell lymphoproliferative disorders (T-CLPD), obtained when detecting increasing numbers of events in the malignant cluster. As shown in Table 2, the CVs of the B-CLL and T-CLPD samples were very close to those predicted by the Poisson distribution. Fifty to sixty events were required to obtain a CV less than 15%. Strikingly, in MM samples, the CVs were around 2

10% regardless of the size of the cluster, even when as few as 20 malignant events were detected (possibly because it is easier to identify malignant cells from MM than from other hematological malignancies, since myelomatous plasma cells usually occupy a space in which background events are scarce). In summary, acquiring 6 million leucocytes is feasible with a digital cytometer on a routine basis. Because detection of 50-60 malignant cells is required to get a CV less than 15%, a sensitivity of 1 " 10-5 is achieved. Being able to apply routinely MRD FC assays with high sensitivity would be very valuable, especially in cases where molecular techniques cannot be used. Esther Domingo 1, Cristina Moreno 1, Alfonso Sánchez-Ibarrola 1, Carlos Panizo 2, José Antonio Páramo 2 and Juana Merino 1 1 Department of Immunology, Clínica Universidad de Navarra, University of Navarra; 2 Department of Haematology, Clínica Universidad de Navarra, University of Navarra References 1. Béné MC, Kaeda JS. How and why minimal residual disease studies are necessary in leukemia: a review from WP10 and WP12 of the European LeukaemiaNet. Haematologica 2009;94:1135-50. 2. Campana D. Minimal Residual Disease in Acute Lymphoblastic Leukemia. Semin Hematol 2009;46:100-6. 3. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008;111:3941-67. 3

4. Paiva B, Vidriales M, Cerveró J, Mateo G, Pérez JJ, Montalbán MA, et al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood 2008;112:4017-23. 5. Krampera M, Perbellini O, Vincenzi C, Zampieri F, Pasini A, Scupoli MT, et al. Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia. Haematologica 2006;91:1109-12. 6. De Tute RM, Jack AS, Child JA, Morgan GJ, Owen RG, Rawstron AC. A single-tube sixcolour flow cytometry screening assay for the detection of minimal residual disease in myeloma. Leukemia 2007;21:2046-9. 7. Rawstron AC, Villamor N, Ritgen M, Böttcher S, Ghia P, Zehnder JL, et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia 2007;21:956-64. 8. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008;93:431-8. 9. Borowitz MJ, Devidas M, Hunger SP, Bowman WP, Carroll AJ, Carroll WL, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study. Blood 2008;111:5477-85. 10. Dworzak MN, Gaipa G, Ratei R, Veltroni M, Schumich A, Maglia O, et al. Standardization of Flow Cytometric Minimal Residual Disease Evaluation in Acute Lymphoblastic Leukemia: Multicentric Assessment is Feasible. Cytometry B Clin Cytom 2008;74:331-40. 4

11. Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L, et al. Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. Haematologica 2009;94:870-4. 12. Clinical Laboratory Standards Institute. Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved guideline-2nd edition document H42-A2. 2007. CLSI/NCCLS, Wayne, Pennsylvania. 5

Table 1. Accuracy of minimal residual disease assessment in samples with infiltration <10-4. A B % MRD by C RQ-PCR F 10 y b D R M % 1 0.1 0.01 0.001 Sample 0.0001 0.0001 0.001 0.01 0.1 1 10 % MRD by RQ-PCR Sample serially diluted 10-fold % MRD by FC 1.5 0.16 0.016 0.001 4.9 0.33 0.025 0.001 Number of acquired leukocytes (millions) MM 1 MM 2 MM 3 MM 4 B-CLL 1 B-CLL 2 B-CLL 3 B-CLL 4 Sample 0.0127 94 Sample diluted 10-fold 0.5 6 0.017 87 0.012 82 0.02 107 0.015 78 0.024 124 0.025 139 0.022 122 0.0012 7 54 0.0017 100 0.0011 8 87 0.0018 99 0.0014 114 0.0022 181 0.0024 151 0.0024 157 A) Minimal residual disease (MRD) levels measured by flow cytometry (FC) or BCR- ABL1 p190 transcripts quantitation by real-time quantitative PCR (RQ-PCR) in serial dilutions of a Philadelphia cromosome-positive B-II acute lymphoblastic leukemia bone marrow sample. MRD results by FC are expressed as percentage of total leukocytes. MRD results by RQ-PCR are expressed as BCR-ABL1 p190 copy number per 100 molecules of GUS. B) MRD levels measured by FC in samples with 10-5 infiltration: MRD + samples with about 0.01% infiltration from 4 patients with multiple myeloma (MM) and from 4 with B-cell chronic lymphocytic leukemia (B-CLL) were assessed. The percentage of MRD for each sample ( Sample ) represents the mean of 5 independent measurements. In each measurement, 0.5 million leukocytes were acquired. These MRD + samples were diluted 10-fold with normal leukocytes in order to obtain tumoral infiltrations in the range of 10-5 ( Sample diluted 10- fold ). The percentage of MRD in diluted samples was measured by acquiring 6 million leukocytes. The number of malignant events detected in each measurement is shown. 6

Table 2. Precision of minimal residual disease assessment according to the number of events in the malignant cluster MM B-CLL T-CLPD Case 1 Case 2 Case 3 Case 4 Case 5 Case 1 Case 2 Case 3 Case 4 Case 5 Case 1 Case 2 Case 3 Case 4 # Cluster events MRD (%) CV (%) 19 0.0127 9 43 0.013 10 94 0.0127 10 30 0.018 10 44 0.017 10 87 0.017 9 25 0.012 10 38 0.011 10 82 0.012 10 39 0.021 8 54 0.02 10 107 0.02 10 22 0.0027 9 42 0.003 10 91 0.003 8 26 0.016 20 36 0.014 19 78 0.015 11 37 0.023 18 60 0.023 14 124 0.024 7 37 0.022 13 55 0.023 9 139 0.025 10 36 0.021 9 62 0.023 12 122 0.022 10 31 0.049 3 55 0.051 7 130 0.05 9 30 0.04 12 50 0.038 12 272 0.04 4 25 0.025 20 47 0.024 13 79 0.026 11 48 0.031 12 86 0.032 8 186 0.032 8 57 0.031 12 106 0.031 8 193 0.03 2 Minimal residual disease (MRD) was measured in samples from 5 patients with multiple myeloma (MM), 5 patients with B-cell chronic lymphocytic leukemia (B-CLL), and 4 patients with T-cell lymphoproliferative disorders (T-CLPD) by detecting three different levels of malignant cells (approximately 20-30, 40-60, and 80-200). Results are presented as the mean of 5 independent trials. The CV was calculated as (standard-deviation/mean) " 100. 7

Online Supplementary Appendix DOI: 10.3324/haematol.2009.018911 Online Supplementary Figure S1. Time-resolved dot plots of CD38 intensity in 6 million bone marrow leukocytes from a representative minimal residual disease-positive (MRD + ) multiple myeloma sample acquired through a live gate drawn on CD38 ++/+++ cells (left), and of CD19 intensity in 6 million peripheral blood leukocytes from a representative MRD + B-cell chronic lymphocytic leukemia sample acquired through a live gate drawn on CD19 + cells (right). In both cases, data derived from several acquisition tubes were appended to the same file using FACSDiva software. The absence of fluctuations of fluorescence intensity between acquisition tubes is illustrated. 8

Online Supplementary Table S1A. Assessment of the percentage of electronic aborts when normal leukocytes were acquired at increasing event rates. Cell Concentration (million cells/ml) Event Rate (events/sec) 0.25 600 0.4 0.5 1200 0.8 1 2250 1.5 2 3800 2.6 4 6300 4.3 5 7500 5.3 Electronic Aborts (%) Increasing event rates were achieved by increasing the concentration of leukocytes in the acquisition tubes. Results are the mean of three independent trials. Based on these results, we considered that 4000 events/sec could be a reasonable flow rate for routine analysis. Online Supplementary Table S1B. Influence of acquisition event rate on minimal residual disease measurements. 2000 (events/sec) Event Rate 4000 (events/sec) MM 0.001 0.0098 0.01 0.01 0.04 0.04 B-CLL 0.01 0.0097 0.01 0.0087 0.006 0.006 Minimal residual disease was measured in samples from 3 patients with multiple myeloma (MM) and 3 patients with B-cell chronic lymphocytic leukaemia (B-CLL), acquired at 1 or 2 million leucocytes/ml and therefore with an event rate of 2000 or 4000 events/sec, respectively. Similar results were obtained with both event rates. 9

A B CD45 APC-Cy7-A -> MM.fcs CD45 APC-Cy7-A -> CD22 APC-A -> CLL.fcs CD22 APC-A -> CD117 APC-A -> MM.fcs CD56 PE-A -> MM.fcs 0 256 512 768 1024 FSC-A -> MM.fcs CD117 APC-A -> CD56 PE-A -> 0 256 512 768 1024 FSC-A -> CD23 PE-A -> CLL.fcs 0 256 512 768 1024 FSC-A -> CLL.fcs CD23 PE-A -> 0 256 512 768 1024 FSC-A -> Online Supplementary Figure S2. The specificity of detection of minimal residual disease of multiple myeloma and B-cell chronic lymphocytic leukemia when a total of 10 million leukocytes are acquired. Panel A: Representative dot plots of 2 million bone marrow leukocytes from a MRD + multiple myeloma (MM) sample (left column) and 10 million normal b o n e marrow l e u k o c y t e s ( r i g h t c o l u m n ) s t a i n e d w i t h CD38/CD56/CD138/CD19/CD117/CD45. Recorded events correspond to gated CD38 +++ cells. The red dots are myelomatous plasma cells (CD38 ++ CD138 ++ CD56 ++ CD19 - CD117 + 10

CD45 - ). The green dots are normal plasma cells (CD38 +++ CD138 ++ CD56 - CD19 + CD117 - CD45 + ). Panel B: Representative dot plots of 2 million peripheral blood leukocytes from a MRD + B-cell chronic lymphocytic leukemia (B-CLL) sample (left column) and 10 million normal peripheral blood leukocytes (right column) stained with CD20/CD23/CD5/CD19/CD22/CD3. The recorded events correspond to gated CD19 + cells. The red dots are B-CLL cells (CD20 d+ CD22 d+ CD5 + CD23 + ). The gray dots are normal B- lymphocytes. No clusters of events mimicking malignant cells were detected in the normal samples. 11