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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From Haematological Malignancy Diagnostic
Service, Leeds General Infirmary, Leeds, United Kingdom;
Nottingham City Hospital, Nottingham, United Kingdom; and the
Sir William Dunn School of Pathology, University of Oxford,
Oxford, United Kingdom.
Previous studies have suggested that the level of residual disease
at the end of therapy predicts outcome in chronic lymphocytic leukemia
(CLL). However, available methods for detecting CLL cells are either
insensitive or not routinely applicable. A flow cytometric assay was
developed that can differentiate CLL cells from normal B cells on the
basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and
can detect one CLL cell in 104 to 105
leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in
nonresponders. Patients with more than 0.01 × 109/L
circulating CLL cells always had significant (> 5%) marrow disease,
and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer
Institute (NCI) criteria, the detection of residual bone marrow disease
at more than 0.05% of leukocytes in 6 out of 25 patients predicted
significantly poorer event-free (P = .0001) and overall
survival (P = .007). CLL cells are detectable at a median
of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable
patients with less than 0.05% CLL cells at end of treatment. All
patients with detectable disease have progressively increasing disease
levels on follow-up. The use of sensitive techniques, such as the flow
assay described here, allow accurate quantitation of disease levels and
provide an accurate method for guiding therapy and predicting outcome.
These results suggest that the eradication of detectable disease may
lead to improved survival and should be tested in future studies.
(Blood. 2001;98:29-35) The goal of conventional therapy for chronic
lymphocytic leukemia (CLL) is to control the disease and rarely results
in the complete eradication of detectable tumor cells.1,2
Even purine analogue therapy, which is the most effective conventional
therapy for inducing complete remission,3 results in a
return of normal polyclonal B lymphocytes in only 40% of
patients.4 This is the principle reason why the assessment
of minimal residual disease (MRD) in CLL has not been considered to be
important and why the criteria for response to therapy have defined
"complete" remissions when there is likely to be a significant
level of disease detectable by modern techniques.5 The
application of novel therapies, such as allogeneic or autologous
hematopoietic stem cell transplantation6 and monoclonal
antibodies,7,8 have resulted in a significant proportion
of patients attaining much more profound responses. There is evidence
to suggest that such responses may be associated with improved
outcomes.9,10 The current development of newer agents, the
potential of immunomodulatory therapies, and the possible combination
of 2 or more of these therapies promises to make the goal of
eradicating detectable disease by the most sensitive techniques a
realistic one.
CAMPATH-1H (alemtuzumab) is a humanized monoclonal antibody specific
for the CD52 antigen. The antigen is expressed on all lymphocytes as
well as monocytes, macrophages, and spermatozoa.11,12 CAMPATH-1H is extremely effective at killing lymphocytes in vitro and
in vivo.11,12 The humanization of the antibody has
abrogated potential human antimurine antibody (HAMA)
responses13-15 and resulted in an agent that is extremely
effective at inducing remission in patients with CLL.8,16
Similarly, treatment with autologous transplantation has been shown to
induce complete remission in a large proportion of
patients.6
Because such therapeutic strategies result in a profound depletion of
CLL cells, effective monitoring of both response and outcome requires
more sensitive methods for the detection of low-level disease. The 2 most effective methods currently used for the detection of MRD in CLL
utilize either flow cytometry or the polymerase chain reaction (PCR).
The majority of flow cytometric analyses depend on the detection of an
excess of CLL cells (> 25% of CD19+ cells being
CD5+) and monoclonality of light chain
expression.10,17-19 Although these techniques are more
sensitive than a morphologic assessment, they are hampered by the
presence of normal B cells. Upper limits for detection of residual CLL
cells have been set according to the normal range for CD5 expression;
however, up to 90% of normal B lymphocytes express CD5 in the
months following transplantation in patients without B-cell
malignancies.20 Conventional flow cytometry is therefore
only suitable when the majority of B lymphocytes are neoplastic. More
recent techniques have used the differential expression of antigens on
CLL cells in comparison with normal B cells, such as
CD79b,21 or CD20.22 However, such flow
cytometric techniques are only informative in a proportion of patients,
because of the interpatient variation in antigen expression.
Although PCR techniques have generally been considered to be more
sensitive than flow cytometric analysis, they can only be applied to
approximately 70% to 80% of patients, as mutations in the
immunoglobulin-H (IgH) gene can abrogate binding of consensus primers.
The use of consensus primers alone to demonstrate clonality is limited
by the presence of normal leukocytes, such that there is a maximum
sensitivity of 1 in 104. In common with conventional flow cytometric
analysis, the detection of CLL cells is also limited by the
presence of normal B cells, such that CLL cells may be detected
when they represent more than 2% of total B cells.23-25
The interference of normal polyclonal B cells can be overcome by
generating primers that are specific to each individual patient (allele-specific oligonucleotide [ASO]-PCR) which are able
to detect a single CLL cell in 106 normal
cells.26 Unfortunately, ASO-PCR is extremely labor
intensive and very expensive. In addition, standard ASO-PCR is not
quantitative and the results are usually not immediately available.
Thus ASO-PCR is not ideally suited for the monitoring and guiding of
therapy. ASO-PCR combined with "real-time" PCR is quantitative but
has a reduced sensitivity detecting a single CLL cell in up to
105 normal leukocytes.27
Major recent advances in flow cytometry, in particular the development
of sequential gating strategies; the improvement in flow cytometer
technology; and the use of multicolor fluorescence permits the analysis
of extremely large numbers of cells and the subsequent identification
of very small populations of cells. This paper describes the
development of a flow cytometry technique able to detect a single CLL
cell in 105 normal cells (MRD Flow). The technique is
quantitative, applicable to all patients with CLL, not affected by the
presence of polyclonal B cells, and can be used in either blood or bone
marrow. We also describe its application in the monitoring of treatment
and prediction of outcome in patients with CLL undergoing therapy with
CAMPATH-1H and/or autologous stem cell transplantation.
Patients
Responses are reported according to the NCI criteria for
response5 except complete remission (CR), which is
separated into 2 categories: MRD+ for patients achieving
the criteria for CR but with CLL cells representing more than 0.05% of
bone marrow leukocytes, and MRD Sample preparation
Flow cytometry A minimum of 0.5 × 106 leukocytes (1.0 × 106 for dilution studies) was incubated with 5µL of each pretitred antibody per 500 000 cells for 20 minutes at 4°C, washed twice, and acquired using a Becton Dickinson (Oxford, United Kingdom) FACSort with CELLQuest v3.1 software. Between 50 000 and 800 000 total cells were analyzed in each test. In all cases, B cells were identified using a sequential gating strategy, setting a region on CD19 (HMDS) versus side scatter (SSC), followed by a light scatter region, and then ensuring that no CD3+ events fell within the combined gate. Dilution studies of B cells into K562 cells showed that this strategy allows detection of one B cell in up to 105 leukocytes with less than 10% coefficient of variation when 5 × 106 total cells were analyzed (data not shown). Antibodies used were CD5, CD10, CD11a, CD23, CD38, kappa and lambda (in-house conjugates), CD20, CD79b (Immunotech, Marseilles, France), FMC7 (Chemicon International, Harrow, United Kingdom), CD22 (Becton Dickinson), and CD79a (Serotec, Oxford, United Kingdom).To allow measurement of the difference in antigen expression between
neoplastic and normal cells while excluding the variation between
antibodies, the Kolmogorov-Smirnov analysis (K-S analysis) was
used.29 This calculates the cumulative proportion at which the maximum difference between 2 histograms occurs (ie, for completely separated histograms, D = 1, whereas for completely overlapping histograms, D = 0) (Figure 1). K-S
analysis was applied to histograms of B cells from each CLL (n = 5)
with each normal case for each antibody (ie, 25 tests per antibody).
The D value was calculated for each test, and these were ranked
according to median values and then by minimum value.
Dilution studies Calibrite beads (Becton Dickinson) were used to allow accurate determination of dilution for sensitivity assessment. Target cells were mixed with an approximately tenfold excess of fluorescein isothiocyanate (FITC) beads, and diluent cells mixed with an approximately equivalent number of phycoerythrin (PE) beads, and the cell-to-bead ratio was calculated for both prior to dilution. Target cells were then diluted into diluent cells, and acquired as above. The excess of FITC beads allows an accurate calculation of actual target to diluent cell ratio, even at high dilutions, and independent of pipetting errors.IgH PCR analysis DNA was extracted and amplified as reported previously23 with a 5' FITC-labeled JH consensus primer and a FR3 consensus primer. Products were then electrophoresed and analyzed using an ABI automated DNA sequencer model 373A (Applied Biosystems, Cheshire, United Kingdom).Immunohistochemistry Sections (3 µm) were stained with MGG, H&E, or CD20. For CD20 staining, 3 µm sections underwent antigen retrieval by microwaving in citrate buffer, (pH 6.0, 8 minutes irradiation, 5 minutes standing, 3 minutes irradiation, then 20 minutes standing). Sections were incubated in CD20 (Dako, Ely, Cambridgeshire, United Kingdom) for more than 1hour, and visualized using a Streptavidin Biotin technique (Dako-Duet K492), with 3,3' diaminobenzidine (Sigma), and counterstained with Harris Haematoxylin.
Identifying antibodies that can distinguish CLL cells from normal B cells In order to detect CLL cells in a minimal disease setting, it is necessary to identify the antibodies that provide complete separation of CLL cells from normal B cells in all patients. Therefore, the expression of a range of antibodies (CD5, CD10, CD11a, CD20, CD22, CD23, CD38, CD79a, CD79b, FMC7, kappa, and lambda) was assessed on presentation CLL cells from 10 patients, and on peripheral blood B lymphocytes from 5 healthy individuals. To allow measurement of the difference between neoplastic and normal cells while excluding the variation between antibodies, Kolmogorov-Smirnov analysis was used. Each antibody showed some degree of overlap between the levels of expression by normal and by neoplastic cells. However, the greatest separation (D value closest to 1) was found for CD20, CD79a/CD79b, and CD5 respectively (Figure 1). These antibodies were unsuitable for bone marrow assessment as B-cell progenitors, which comprise up to 90% of B cells in posttransplant or post-CAMPATH-1H patients, have weak or no expression of CD20 and CD79. We therefore studied 3 antigens CD34,
CD10, and CD38that are expressed during the early stages of B-cell
differentiation, but are weak or negative on presentation CLL cells.
Separation of CLL cells from normal B progenitors was again assessed
using Kolmogorov-Smirnov analysis, and CD38 provided significantly
better separation than CD34 or CD10 (n = 35, P < .0001,
Wilcoxon signed rank test).
Determination of antibody combinations that provide resolution of CLL cells from B cells in peripheral blood and bone marrow Because single antigens were not sufficient to allow discrimination of normal B cells from CLL cells, it was necessary to identify combinations of antibodies that would reproducibly separate these populations. The ability of antibody combinations to resolve CLL cells from normal B cells was assessed by setting regions around the CLL cells identified by 2 markers (eg, CD5 vs CD79b, CD38 vs CD79b, etc) such that the regions contained more than 95% neoplastic cells. The regions were then applied to normal samples, and the percentage of B cells that fell within each region was calculated. If less than 2% of normal B lymphocytes fell within a particular region, that region was considered adequate to resolve CLL cells from normal cells for that patient. This was performed for 17 sets of CLL cells against peripheral blood B cells from 5 healthy individuals, and bone marrow B cells from 3 healthy individuals.In peripheral blood, a combination of CD19, CD5, CD20, and CD79b was
sufficient and necessary to discriminate CLL cells from normal B cells
in all the cases tested. In bone marrow, a combination of CD19/5/20/79b
was effective in 14 out of 17 cases, CD19/5/38/79 in 14 out of 17 cases
and CD19/5/38/20 in 11 out of 17 cases. Of these 17 patients, 8 showed
CD38 expression according to the criteria reported
previously.30 In all of these cases, there was no overlap
between CLL cells and B progenitors, as the latter express very high
levels of CD38. Using all 3 combinations allowed discrimination of CLL
cells from normal B cells in all cases (Figure 2).
Specificity and sensitivity of MRD Flow compared with conventional flow cytometry and IgH-PCR Serial dilution studies were performed to determine the specificity and sensitivity of the MRD Flow assay. CLL cells from 3 patients were mixed with normal leukocytes in serial 4-fold dilutions from 1:1 to 1:16 384. Three aliquots, each of one million cells, were prepared: one was stained with CD19/CD5/CD20/CD79b, one with CD19/CD5/kappa/lambda, and IgH-PCR was performed on DNA extracted from the remaining aliquot.In addition to the PCR results, the detection of CLL cells was compared using 4 different flow cytometric gating strategies. These were (1) 2-color analysis of CD19 and CD5 coexpression, in which CLL cells are considered to be present if more than 25% of CD19+ B cells coexpress CD517-19; (2) 3-color analysis of light chain restriction by CD19+ cells, in which CLL cells are considered to be present if more than 75% of B cells express kappa or lambda; (3) 4-color analysis of light chain restriction within the CD19+CD5+ fraction, in which CLL cells are considered to be present if more than 75% of CD5+ B cells express kappa or lambda; and (4) 4-color MRD Flow analysis as described above. The results are shown in Table 1, with
the dilution curves shown in Figure 3.
Analyzing light chain expression simultaneously with CD19 and CD5
coexpression results in a modest improvement in sensitivity over
analysis of CD19 and CD5 alone. However, the use of the 4-color MRD
Flow technique results in a 2-log increase in sensitivity in comparison
with conventional 4-color analysis, despite identical methods for
identification of total B cells. This was because the MRD Flow assay
has a much higher specificity for identification of CLL cells than
conventional flow methods. The conventional assay could detect an
abnormal kappa:lambda ratio in the CD5+ B cells when CLL
cells represented approximately 30% of B cells, whereas MRD Flow could
consistently detect CLL cells when they represented more than 1% of B
cells. Enumeration of CLL cells by conventional techniques became
increasingly inaccurate at the limits of detection because of the
number of normal CD19+5+ B cells present. Thus
the numbers of CLL cells observed using conventional flow were between
2- and 60-fold greater than the actual numbers (ie, 100%-6000%
error). In contrast, the error between observed and actual CLL cell
numbers identified by MRD Flow was on average less than 25%, and
always less than 50% even at the limits of detection.
MRD Flow was also at least 2 logs more sensitive for the detection of CLL cells than Fr3-primer IgH-PCR. Although we have previously demonstrated that this test can detect CLL cells when they represent as few as 2% of B cells, the primers also bind germline sequences in non-B cells. Although these sequences do not undergo logarithmic amplification during the PCR reaction, their presence limits the sensitivity of detection. Thus the sensitivity of consensus-primer IgH-PCR was equivalent to that of conventional flow in the presence of excess leukocytes, and the test could not detect CLL cells when they represented less than 0.4% of leukocytes. In bone marrow aspirate samples from 37 patients, CLL cells were detected by MRD Flow cytometry in 22% (8/37) PCR-negative samples, whereas all 29 PCR-positive samples also had detectable CLL cells by MRD Flow. There were no samples in which CLL cells were detected by IgH-PCR but not by MRD Flow. In keeping with the dilution study results, PCR-negative samples contained less than 0.2% CLL cells, which were readily identified by MRD Flow. Thus MRD Flow is more sensitive than previously identified flow cytometry assays as well as consensus Fr3 primer IgH-PCR. CLL cells were reproducibly detected when they represented 0.01% of leukocytes or 2% of B cells. Detection of CLL cells in patient material: comparison with trephine biopsies Detection of CLL cells by MRD Flow was compared with morphologic assessment of trephine biopsy in 103 samples. In general, there was good correlation between the extent of infiltration on the trephine biopsy and the percentage of CLL cells detected by MRD Flow (Figure 4). CLL cells were detected by MRD Flow (ie, > 0.05% of leukocytes) in 32% (8/25) of samples with no morphologic evidence of tumor.
The potential for sampling error in aspiration Monitoring disease levels: comparison between peripheral blood and bone marrow during and after therapy Patients undergoing CAMPATH-1H therapy show clearance of peripheral blood disease even if bone marrow infiltration or lymph node size are not altered.16 More sensitive analysis of the peripheral blood demonstrates that in the majority of patients, circulating CLL cells levels fall to below 0.01 × 109/L. This includes patients who show little or no alteration in the level of bone marrow disease: 82% (36/44) of samples with over 30% marrow infiltration had less than 0.01 × 109/L circulating CLL cells. Therefore, for guiding therapy, a bone marrow assessment may not be indicated until the circulating CLL count drops below 0.01 × 109/L.In contrast, there was a direct correlation between circulating and
bone marrow disease levels in patients more than 3 months after therapy (Figure 5). If the
peripheral count was more than 1.0 × 109/L, virtually
all patients (33/35) had more than 30% bone marrow CLL cells. This
suggests that a peripheral count above 1.0 × 109/L may
provide an optimal time point to consider bone marrow examination and
possible retreatment. In 14 patients with fewer than
0.01 × 109/L circulating CLL cells at least 3 months
after treatment, only 2 patients had more than 0.05% bone marrow CLL
cells (0.11% and 0.12% of total leukocytes respectively). Thus, the
level of peripheral disease in patients more than 3 months after
treatment can provide a good indication of the degree of bone marrow
involvement.
Predicting outcome according to residual disease levels The MRD Flow assay provides one of the most sensitive methods of assessing disease levels in patients with CLL. However, it is not clear whether the application of a sensitive residual disease assay can provide a more accurate prediction of outcome than the current NCI criteria. In order to assess this, we have compared the event-free and overall survival for patients achieving a complete remission by NCI criteria after CAMPATH-1H and/or autologous transplantation with or without detectable residual disease. Patients were defined as MRD+ if CLL cells represented more than 0.05% of bone marrow leukocytes at 3 months after treatment, or MRD if
there were less than 0.05% CLL cells present. Of 7 previously untreated patients receiving fludarabine and autologous PBSCT, all
became MRD after receiving a transplant. Of 10 pretreated
patients receiving CAMPATH-1H alone, 8 out of 10 became
MRD whereas 2 out of 10 had detectable disease at the end
of therapy. Of 8 pretreated patients receiving CAMPATH-1H followed by
autologous PBSCT, 4 out of 8 became MRD whereas the
remaining 4 out of 8 had detectable disease at end of therapy.
Overall, 19 (76%) out of 25 were MRD
Application of MRD Flow to monitoring patients after therapy Patients achieving MRD status after treatment have a
prolonged event-free survival, but it is not clear whether any of these patients are being `cured.' In order to test this, patients were monitored sequentially to determine whether early signs of relapse could be identified, and if so whether the kinetics of disease progression could be determined.
Return of CLL cells has been detected in 9 out of 18 evaluable patients
initially identified as MRD All patients with detectable CLL cells show a steadily increasing disease levels with time, even in patients with low initial levels of disease. The median doubling time was 121 days (range, 54-244 days) in 6 patients assessed. As the doubling time is highly variable, prediction of time to clinical progression requires knowledge of both proliferation rate and initial disease levels. With close monitoring, it is possible that MRD Flow could be used in patients with subclinical levels of CLL to allow prediction of time to disease progression.
In this study, we report the development of a flow cytometric assay (MRD Flow) based on the differential expression of antigens by CLL cells compared with normal B cells. The assay has sensitivity comparable with the most sensitive techniques currently available (eg, ASO-PCR) but is readily quantitative and rapid (results may be generated within 1 hour). As such, the technique may be used to guide therapy. In contrast to IgH-PCR techniques, MRD Flow is applicable to all patients assessed so far, and at all stages of disease as no prior knowledge of the presentation phenotype is necessary. There are clear advantages to MDR Flow over previous flow-cytometric assays: those that consider only CD5/CD79b21 or CD5/CD2022 cannot be used to monitor all patients, whereas those that use light-chain restriction are insensitive in the presence of polyclonal B cells. We have used this assay, as well as standard criteria, to monitor response and follow-up of patients with CLL treated with CAMPATH-1H and/or autologous PBSCT. The dynamics of response to CAMPATH-1H were varied with an immediate clearing of CLL cells from the peripheral blood in almost all of the patients but with a slower clearance of the bone marrow. Analysis of peripheral blood levels during CAMPATH-1H therapy could not predict the level of bone marrow disease, as circulating disease is rapidly depleted. As such, the peripheral blood CLL cell levels could be used to predict the most appropriate time to perform bone marrow analysis, which is not necessary until circulating disease is below 0.01 × 109/L. The dynamics of blood and bone marrow response to other agents is not clear, and requires further study. Once off treatment, the levels in the peripheral blood did provide a more direct indication of bone marrow involvement. Patients with less than 0.01 × 109/L circulating disease always had less than 1% CLL cells in the bone marrow. In contrast, patients with more than 1 × 109/L blood CLL cells almost always had more than 30% bone marrow disease. The use of MRD Flow demonstrates that approximately a quarter of the
patients who achieve a complete remission by NCI criteria after
CAMPATH-1H and/or autologous transplantation had detectable disease
(MRD+) whereas the remainder had no detectable disease
(MRD The durability of response to CAMPATH-1H is varied, but patients
who achieve an MRD In summary, overall and progression-free survival are dependent
on both the depth of remission and the CLL doubling time, but
calculation of these values requires highly sensitive and quantitative
techniques. MRD+ patients show inevitable disease
progression, whereas MRD
We thank Millenium and Ilex Partners and the Therapeutic Antibody Centre, Oxford, United Kingdom, for provision of the CAMPATH-1H used in this study.
Submitted August 1, 2000; accepted February 28, 2001.
Supported by the Leukaemia Research Fund, London, United Kingdom and Yorkshire Cancer Research, Harrogate, United Kingdom.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andy Rawstron, HMDS, Algernon Firth Bldg, Leeds General Infirmary, Leeds LS1 3EX, United Kingdom; e-mail: andy.rawstron{at}newscientist.net.
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J. Esteve, N. Villamor, D. Colomer, E. Montserrat, A. C. Rawstron, and P. Hillmen Different clinical value of minimal residual disease after autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia Blood, March 1, 2002; 99(5): 1873 - 1874. [Full Text] [PDF]
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K. R. Rai, H. Dohner, M. J. Keating, and E. Montserrat Chronic Lymphocytic Leukemia: Case-Based Session Hematology, January 1, 2001; 2001(1): 140 - 156. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) for the
presence of CLL cells at each dilution for the different methods of
assessing residual disease. Criteria for detection of CLL cells were as
follows: (1) IgH-PCR: there is at least 2-fold greater PCR product with
the same size as the CLL cell rearrangement product than would be
expected for a normal distribution
