Analysis of Residual Disease in Chronic Lymphocytic Leukemia by Flow

Analysis of residual disease in chronic lymphocytic leukemia by ﬂow cytometry E Cabezudo, E Matutes, M Ramrattan, R Morilla and D Catovsky

Academic Department of Haematology and Cytogenetics, The Royal Marsden Hospital and Institute of Cancer Research, London, UK

We have investigated the value of both conventional and quan- Material and methods titative flow cytometry to detect minimal residual disease in 21 CLL patients in remission including bone marrow histology: Patients eight in complete remission (CR), 11 in nodular partial remission (nPR) and two in PR. The techniques used were dou- Twenty-one patients with B cell chronic lymphocytic leu- ble immunostaining with CD5 and CD19 and quantitative esti- kaemia (CLL) were investigated. The diagnosis was based on mation of the number of both antigens with standard 24,25 microbeads. Reference values were established on normal per- clinical, morphologic and immunologic criteria. Patients ipheral blood and bone marrow controls. Patients were con- were studied prior to treatment and at the time of maximal sidered in ‘immunological’ remission when the percentage of response which was defined as: CR, nodular PR (nPR) or PR. CD5+ CD19+/total CD19+ cells was Ͻ25% in PB and Ͻ15% in BM. Criteria for CR were: normal physical examination, absence of In six of the eight patients in CR, CLL cells were still detectable constitutional symptoms, peripheral blood lymphocyte count by flow cytometry. Only two patients, that underwent allogeneic р4 × 109/l, platelets Ͼ100 × 109/l, Hb Ͼ11 g/dl and Ͻ30% bone marrow transplant, achieved immunological remission. CLL samples showed significantly higher CD5 and lower CD19 lymphoid cells in the bone marrow (BM) aspirate with normal antigen density than normal controls (P Ͻ 0.001). Persistence histology. The presence of residual nodular or interstitial of residual disease was a predictor of time to progression. lymphoid aggregates in the bone marrow biopsy was defined None of the two patients in immunological remission relapsed as nPR. The presence of more than 30% lymphocytes in the within a period of 13 and 33 months, whilst two of the six bone marrow aspirate after normalization of all the other dis- patients in CR with positive flow cytometry relapsed 3 and 6 ease features was defined as PR. Relapse was defined as an months after achieving CR. This study demonstrates that flow × 9 cytometry contributes to increase the sensitivity of the clin- increase in the absolute lymphocyte count above 5 10 /l icohematological criteria to detect residual malignant cells in and/or reappearance of abnormal physical findings. The clini- CLL patients and may be useful to monitor disease status cal and hematological data of the patients pre- and post-ther- following treatment. apy are shown in Table 1. Mean age was 56 years (range, Keywords: chronic lymphocytic leukemia; minimal residual dis- 31–75 years). Seven patients were already in PR before they ease; quantitative flow cytometry received treatment prior to evaluation (Table 1). Two patients underwent allogeneic bone marrow transplantation (Nos 2 and 4) and patient No. 6 underwent a matched unrelated allo- Introduction geneic BMT. Two patients in CR after BMT remained with persistent thrombocytopenia and/or anemia. Following vari- The introduction of new therapeutic agents and strategies for ous therapeutic regimens, eight patients achieved CR (Nos 1– the treatment of CLL has increased the proportion of patients 8), 11 nPR and two PR (Nos 9–21). 1–8 that achieve complete remission (CR). This may result in Eleven peripheral blood and 10 bone marrow samples from 9,10 changes in the natural history of the disease. The possibility healthy donors (age 23–50 years) were used to establish refer- of achieving CR has increased the need for developing accur- ence values for the percentage of CD5+ B lymphocytes and ate evaluation methods to monitor response to therapy as this the quantitative expression of CD5 and CD19. may have management implications.3,11,12 Flow cytometry has been widely used to detect minimal residual disease (MRD) in acute leukemia13 and only more recently in chronic lymphoid Immunostaining disorders.11,14,15 In all these studies, the detection of the Mononuclear cells were isolated from fresh heparinized per- residual malignant population by flow cytometry is mainly ipheral blood and bone marrow samples by density gradient based on the presence of aberrant phenotypes and/or quanti- centrifugation with Lymphoprep (Nycomed, Oslo, Norway) tative abnormalities of antigen expression.16–21 and washed three times in Hank’s balanced salt solution. A CLL has a distinct B cell marker profile characterized by total of 1 × 106 cells were used per test. Fifty microliters of 2% weak surface immunoglobulin and expression of CD5.22 The AB serum was added to each tube prior to incubation with CD5 antigen is only expressed in a small percentage of normal the monoclonal antibody (McAb). Double-labelling immuno- blood and bone marrow B lymphocytes,23 therefore the staining was performed using CD5-FITC and CD19-RD1, for phenotype of CLL can be exploited to detect MRD. We have 15 min at room temperature. McAbs of the same IgG subclass investigated the clinical significance of detecting MRD in 21 were used as negative controls in all the experiments. After patients with CLL in CR or partial remission (PR) by using dou- incubation, cells were washed three times with phosphate- ble labelling with CD5/CD19 and estimating quantitatively the buffered solution (PBS)/azide, resuspended in 0.5 ml Isoton number of molecules per cell. (Coulter, Hialeah, FL, USA) and analyzed on the flow cytometer.

Assessment of antibody binding capacity (ABC) by Correspondence: D Catovsky, Academic Department of Haematology Quantum Simply Cellular method and Cytogenetics, The Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK This was performed on mononuclear cells from fresh peri- Received 29 January 1997; accepted 18 July 1997 pheral blood (10 patients and 11 controls) and from cryo- Analysis of residual disease in CLL by ﬂow cytometry E Cabezudo et al 1910 Table 1 Clinical and hematological data in 21 CLL patients pre- and post-treatment

Patient Age/ Pre-treatment Treatment Post-treatment Sex Stage PB BM Hb Platelet PB BM BM MRD Status Time to Follow-up lymphocytes trephine (g/dl) (109/l) lymphocytes trephine lymphocytes (ﬂow) relapse (months) (109/l) (109/l) (%) (months)

1 75/M B 57.2 Diffuse Fludarabine 15.1 102 0.37 Normal 4 Y CR 0 7 2 55/F A 2.49 Interstitial BMT 14.9 305 3.9 Normal 21 N CR 0 33 3 48/F B 154.8 ND Fludarabine 13.7 174 1.7 Normal 12 Y CR 0 5 4 50/M B 0.83 Normal BMT 10.7 78 0.69 Normal ND N CR 0 13 5 50/M B 2.4 Nodular Campath 14.7 237 4 Normal 10 Y CR 3 7 6 42/M B 3.1 Interstitial BMT 10.2 224 0.93 Normal 6 Y CR 0 3 7 48/M A 7.6 Interstitial Campath 13.6 222 0.35 Normal ND Y CR 6 11 8 51/M A 4.1 Nodular Campath 11 207 2.3 Normal 15 Y CR 0 7 9 64/M B 59.6 Diffuse Epi/Cloram 11.7 197 3.62 ND 42 Y PR 12 20 10 66/M B 5.48 ND Cloram 14.3 155 2.05 Nodular 20 Y nPR 7 16 11 31/M A 1.39 Interstitial Campath 12.15 107 0.54 Interstitial 9 Y nPR 0 4 12 60/M B 180 Diffuse HDMP 13.9 116 4 Diffuse 40 Y PR 0 3 13 51/M B 3.4 ND Campath 15.4 288 1.26 Nodular 6 Y nPR 6 9 14 63/M C 50.9 Interstitial Fludarabine 13.3 166 1.4 Interstitial ND Y nPR 0 2 15 65/M B 3.7 Interstitial HDMP 14.8 119 3.1 ND ND Y nPR 0 2 16 57/M B 56.4 Diffuse Fludarabine 11.8 135 1.4 Interstitial 11 Y nPR 0 4 17 56/M C 4.6 ND Fludarabine 14.3 452 1.1 Interstitial 9 Y nPR 26 31 18 64/M A 130 ND Fludarabine 13.9 178 2.8 ND ND Y nPR 24 31 19 66/F B 224.6 ND Fludarabine 15.3 269 2.6 Nodular 12 Y nPR 14 28 20 72/M B 27.1 ND Fludarabine 13.9 214 1.6 Interstitial ND Y nPR 0 18 21 53/F C 911 ND Fludarabine 14.1 104 1.34 Interstitial 14 Y nPR 0 5

BMT, allogenic bone marrow transplant; Epi/Cloram: epirubicin/clorambucil; HDMP: high-dose methylprednisolone; CR: complete remission; nPR: nodular partial remission; PR: partial remission; MRD (ﬂow), minimal residual disease detected by ﬂow cytometry; ND, not done; Y, yes.

preserved cells (five patient samples and five controls). Cell Statistical analysis viability, assessed by Trypan blue exclusion, was always greater than 80%. Fifty microliters of microbeads per test were Results were expressed as mean ± standard deviation (s.d.) stained and processed in parallel with the cell samples using and compared with the Student’s t-test. The Spearman rank the same amount of reagent, incubation time (1 h, as rec- correlation test was used to compare the CD5 and CD19 ABC ommended by the manufacturer) and method, as described values with the percentage of double positive CD5/CD19 above for the double labelling. cells. Quantum Simply Cellular Microbeads kit (Sigma, St Louis, MO, USA) provides a method for the evaluation of the number of molecules per cell by flow cytometry. The microbeads lab- Results elled with a goat anti-mouse polyclonal Ig provide standards to calibrate the fluorescence scale of the flow cytometer in Immunophenotyping units of antibody binding capacity (ABC), converting the mean fluorescence intensity (MFI) into number of antigen molecules per cell.26 The methodology applied has been previously described by Farahat et al.27 The QuickCal (Sigma) software Normal samples: Results of the analysis of normal samples is designed to construct a calibration curve for each specific are shown in Table 2. Based on these data, a percentage of reagent and derive ABC values for the cells under study. CD19+ CD5+ double positive lymphocytes out of the CD19+ lymphocytes (CD5+ CD19+/CD19) greater than 25% in PB and greater than 15% in the BM (mean ± 2 s.d.) were regarded as Flow cytometer analysis abnormal. Immunological remission was defined by the percentage of CD5+ CD19+ over the percentage of CD19(B) lym- Acquisition and analysis of data were performed on a FAC- phocytes in PB and/or BM below the normal range. Scan flow cytometer with LYSYS II software (Becton Dickin- son, San Jose´, CA, USA). Standard microbeads labelled with Table 2 Immunological evaluation in normal controls CD4-FITC and CD8-PE were used to set the compensation. For each sample 104 cells were acquired. A second acqui- Sample CD19+ CD5+CD19+ CD4+CD19+/ sition gating on the CD19 positive population was performed (%) (%) CD19 (%) in all cases with low CD5/CD19 double positive population as well as in the controls. Acquisition was stopped when 104 PB (n = 11) 3.7 ± 1.8 0.4 ± 0.4 10 ± 6 CD19 cells had been collected or all cells in the tube had (range) (1.4–7) (0.04–1.5) (5–25) BM (n = 10) 14.5 ± 9.5 1 ± 0.8 6 ± 4 been analyzed. Analysis was performed in the lymphocyte (range) (5.4–37.5) (0.1–2.3) (1–15) area gated on the forward and side scatters. The monoclonal antibodies used in this study were CD5- PB, peripheral blood; BM, bone marrow. FITC (UCH-T2) (Sera-Lab, Loughborough, UK), CD19 (B4- CD19+ and CD5+ CD19+ refer to the lymphocyte gate in FSC/SSC. RD1) (Coulter Clone), CD4-FITC (Edu-2) (Sera-Lab) and CD8- CD5+CD19+/CD19 refers to the total CD19 lymphocytes. Results are PE (Leu-2a) (Becton Dickinson). expressed as mean ± standard deviation. Analysis of residual disease in CLL by flow cytometry E Cabezudo et al 1911 Table 3 Immunological evaluation in CLL patients in CR CD5 and CD19 antigen density in normal controls

Patient Sample Time CD5+ CD19 CD5+CD19+/ + The quantitative evaluation of CD5 and CD19 in normal (months) CD19 (%) (%) CD19 (%) samples is shown in Table 5 and illustrated in Figure 1a. The analysis of CD5 and CD19 densities in BM samples by quanti- 1 PB 3 1.8 7.3 26 tative flow cytometry was not used in this study due to the 2a PB 32 0.6 5.6 10 2b BM 32 0.6 7.3 8 wide range of distribution of the reference values (data not 3PB52450shown). No statistically significant differences were found 4 PB 10 2 27 7 between fresh and cryopreserved peripheral blood samples for 5 PB 3 5.5 8 68 the assessment of CD5 and CD19. 6a PB 3 0.7 0.8 87 6b BM 3 1.4 2.5 8 7a PB 4 39.7 42 94 7b BM 4 10 36.7 27 CD5 and CD19 antigen density in CLL 8a PB 2 9 12.4 72 8b BM 2 4 21.7 18 The mean number of CD5 molecules per cell was significantly higher in CLL samples than in normal B lymphocytes (Table 5) PB, peripheral blood; BM bone marrow. (Figure 1d) (P Ͻ 0.001). When considering a cut-off point of CD19 and CD5+CD19+ refer to the lymphocyte gate in FSC/SSC; 15 × 103 molecules per cell (the maximum value of CD5 in CD5+CD19+/CD19 % refers to the total CD19 lymphocytes. Time, normal B cells), 77% of the CLL samples had values above months from remission to sample collection. this level (Figure 2). The expression of CD19 was significanly lower in CLL than in normal CD5+ B lymphocytes (P Ͻ 0.001). 72% of CLL samples had values below 10 × 103 molecules per cell (the minimum normal value) (Figure 3). The two Patient samples patients (Nos 2 and 4) in immunological remission had ABC

+ + values for CD5 and CD19 similar to the normal controls Before therapy, the mean percentage of CD5 CD19 lympho- (Figure 1b). In patient No. 6 (sample 6b) residual CLL cells cytes was 60 ± 32 (range 6–98) in PB and 40 ± 30 (range 0.3– + could be identified by their high expression of CD5 compared 80) in the BM. The corresponding mean percentage of CD5 to the normal B cells present in the same sample (Figure 1c), + ± CD19 /CD19 lymphocytes was 91 12 (range 50–100) in PB although the results are not expressed as ABC values. and 85 ± 16 (range 49–100) in the BM. The results of the immunophenotypic analysis post-treatment are shown in Table 3 for patients in CR and in Table 4 for patients in nPR Comparison between CD5+ CD19+ and CD5+ and PR. CD19+/CD19 percentages in the evaluation of After therapy only two patients (Nos 2 and 4) who minimal residual disease underwent BMT were in immunological remission, patient No. 6 still had residual CLL cells in peripheral blood 3 months + + + + Defining a CD5 CD19 cut-off value of 1.2% in PB and 2.3% after the BMT, whilst the proportion of CD5 CD19 /CD19 in the BM (mean ± 2 s.d. of the normal controls), five patients lymphocytes was normal in a BM sample taken at the same (Nos 2, 4, 6, 11 and 20) would be considered in immunolog- time. Flow cytometry suggested that there was residual disease ical remission, including two nPR patients. Expressing the per- in six patients in CR (Table 3) and confirmed persistence of centage of residual CLL cells out of the total B (CD19+) lym- disease in all nPR and PR cases (13 patients) (Table 4). phocytes (gating the B cells by sized scattered/CD19), has one major advantage as results expressed as a percentage of the total lymphoid cells gated by FSC/SSC characteristics permits the acquisition of greater numbers of B cells for analysis. By Table 4 Immunological evaluation in CLL patients in nPR and PR using this method only two patients (Nos 2 and 4) rather than Patient Sample Time CD5+CD19+ CD19 CD5+CD19+/ five were in immunological remission. (months) (%) (%) CD19 (%)

9PB12373897Correlation with outcome 10 PB 4 13.7 16 85 11 BM 0 0.2 0.6 33 With a median follow-up of 6 months (range, 2–33), eight 12 PB 3 24 25 97 patients have relapsed and these included: two CR patients, 13 PB 2 10 11 90 14 PB 2 18 18.2 98 Table 5 Mean ABC (antibody binding capacity) values 15 PB 0 12 12.5 96 (×103) ± s.d. in normal peripheral blood B lymphocytes and CLL 16a BM 1 19.4 22 88 samples 16b PB 3 2.6 3.4 67 17 PB 14 11.4 22 51 Antigen Normal CLL MRD+a MRD−a 18 PB 16 15 20 75 = = = = 19 PB 5 4.6 10.9 42 (n 11) (n 16) (n 8) (n 2) 20 PB 8 1.1 2 55 21 PB 5 52 53 99 CD5 10 ± 223±11 27 ± 11 12 ± 3 (range) (6–15) (10–47) (9–47) (9–14) ± ± ± ± PB, peripheral blood; BM, bone marrow. CD19 14 26392192 CD19 and CD5+ CD19+ refer to the lymphocyte gate in FSC/SSC; (range) (10–17) (4–13) (7–15) (18–21) CD5+ CD19+/CD19 refers to the total CD19 lymphocytes. Time, months from remission to sample collection. aMRD, minimal residual disease by ﬂow cytometry. Analysis of residual disease in CLL by ﬂow cytometry E Cabezudo et al 1912

Figure 1 Double labelling with CD5-FITC/CD19-RD1 in peripheral blood B lymphocytes, gated by CD19/SSC characteristics. (a) Normal plot showing a CD5 weak expression in normal B lymphocytes difﬁcult to differentiate from the CD5 negative B population (ABC for CD5: 9 × 103). (b) Patient in immunological remission after BMT showing a similar pattern to normal control (ABC for CD5: 10 × 103). (c) Patient in CR with residual neoplastic cells as shown by a higher density of CD5 expression compared to (a) and (b) (ABC for CD5: 19 × 103). (d) Patient with active CLL showing 99% cells coexpressing CD5 and CD19 with a pattern of staining similar to (c) (ABC for CD5: 20 × 103).

five nPR patients and one PR. The two patients in immunolog- tive in detecting MRD and offers the advantages of low cost, ical remission continue with no evidence of relapse at 13 and speed and absence of false positive results. Because CLL cells 33 months after BMT, whilst two of the remaining six CR coexpress CD5/CD19, double immunostaining and quantitat- patients with MRD by flow cytometry relapsed. The third BMT ive flow cytometry can exploit the differences in expression patient (No. 6) with residual disease by flow cytometry was of these two membrane antigens by comparison with nor- in CR 3 months after the procedure. All patients are alive at mal samples. the time of this report. There have only been few studies documenting MRD by flow cytometry in CLL, with the number of cases in immunological remission ranging from 10 to 80%. The techniques Discussion used included double labelling with CD5 and a panB McAb and/or assessment of the K/L ratio.11,14,15,30 Differences among There have been a number of reports investigating MRD in these studies might be related to: (1) variability of the criteria CLL, either by molecular or flow cytometry techniques. The to define CR, as some of the reports did not include BM level of detection of residual leukemic cells using molecular histology; (2) variable criteria defining ‘immunological techniques ranges from 1 to 0.0001% depending whether remission’; and (3) sensitivity of the method. In our study, we Southern blot or the polymerase chain reaction (PCR) are have shown that all cases in nPR and PR and six out of eight applied.12,28 The level of detection by flow cytometry has patients considered in CR by bone marrow biopsy did have improved from 1%, if only clonality assessment is carried out leukemic cells detectable by flow cytometry. Although we by the K/L ratio, to 0.001% if quantitative analysis is perfor- only investigated 10 out of the 21 patients by the quantitative med.14,15,29 Therefore flow cytometry appears to be very sensi- method, there was good correlation overall between these Analysis of residual disease in CLL by flow cytometry E Cabezudo et al 1913 Quantitative evaluation of BM samples seems to be less useful than that of peripheral blood due to the wide distribution of values founded in normal controls. This may relate to the staining with CD19 of mature lymphocytes and immature precursors.17 Selection of the B cell population with a McAb not present in B precursors might overcome this problem. Our study has also shown that persistence of MRD assessed by flow cytometry may predict time to progression. Neither of the two patients in immunological remission relapsed within a follow-up of 13 and 33 months, whilst two of the remaining six CR patients who had positive flow cytometry relapsed. BMT seems to be the only procedure, according to our results, that can achieve immunological remission. Differences in overall survival are however difficult to evaluate due to the short follow-up in the majority of series, including the present one. Following BMT (autologous or allogeneic) for the treatment of hematological and non-hematological malignancies, most circulating B cells have been reported to express CD5, and Figure 2 Distribution of antibody binding capacity (ABC) values remained the predominant B cell subpopulation for several for CD5 in normal peripheral blood, CLL patients with residual disease weeks.23 Quantitative analysis (with or without the use of stan- by flow cytometry (CLL) and CLL patients in immunological CR (CLL- dard microbeads) may have a useful role in identifying ICR). Each circle represents a different sample. residual leukemic cells in CLL patients after BMT, that could not be achieved with conventional flow cytometry as shown in one of the patients (No. 6). In conclusion, we have demonstrated here that both double labelling with CD5/CD19 and quantitation of these antigens by flow cytometry are sensitive methods for detecting MRD in CLL, are useful to monitor disease status and therefore to assess the effectiveness of therapeutic strategies.

Acknowledgements

We are grateful to Mr R A’Hern for the statistical analysis. EC had a fellowship from the Spanish Government (FIS). This work was partly supported by grants from the Medical Research Council and the Kay Kendall Leukaemia Fund.

References

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