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Response Assessment in Chronic Lymphocytic Leukemia After Fludarabine Plus
Prednisone: Clinical, Pathologic, Immunophenotypic, and Molecular Analysis
By L.E. Robertson, Yang 0. Huh, James J. Butler, William C. Pugh, Cheryl Hirsch-Ginsberg, Sanford Stass,
Hagop Kantarjian, and Michael J. Keating
The goals of this study were t o evaluate the response t o
treatment in chronic lymphocytic leukemia (CLL) according
t o clinical, pathologic, immunophenotypic, and molecular
features, as well as t o address the clinical significance of each
finding. One hundred fifty-nine CLL patients with either
advanced Rai stage 111 or IV (81 patients) or progressive Rai
stage 0 t o II (78 patients) were treated with fludarabine (30
mg/m2/d intravenously every day for 5 days) plus prednisone (30 mg/m2/d orally daily for 5 days). Thirty-six
patients were previously untreated. The response rates were
12% complete response (CR), 30% nodular complete response (nCR), and 18% partial response (PR). In all patients
who achieved a complete response (both CR and nCR) less
than 30% of nucleated cells were lymphocytes on marrow
aspirate differential analysis; however, nCR patients had
residual nodular and/or interstitial lymphocyte involvement
on marrow biopsy examination. There was no evidence of
leukemic infiltration on marrow biopsy examination in CR
patients. With a median follow-up of 35 months, comparison
of time t o progression in the CR and nCR groups at 2 years
showed a projected 87% versus 55% progression-free survival (P < .03). Residual disease assessment by flow cytometry using simultaneous dual-color staining on blood and
marrow lymphocytes was also performed on each patient.
Residual disease was determined by the expression of CD5
on B lymphocytes and the monoclonality of surface lightchain expression. After six courses of fludarabine plus prednisone, no residual disease was detected by flow cytometry
in 89% of the CRs, 51% of the nCRs. and 19% of the PRs.
Clinical residual disease in PR patients with no residual
disease detectable by flow cytometry was limited t o lymphadenopathy. Time t o progression at 2 years was longer in CR
and nCR patients having no residual disease detected by flow
cytometry (84% Y 39% 2-year progression-free survival,
P < .001). Posttreatment l g gene rearrangement analysis
using JH, JK, and CA probes demohstrated no rearranged
bands and a return t o the germline configuration in five of
seven CRs and two of eight nCRs studied. The molecular
studies were concordant with the dual-parameter immunophenotype results and none of the patients who reverted
t o a germline DNA pattern after treatment have experienced
relapse. The absence of detectable minimal residual disease
by bone marrow biopsy, dual-color flow cytometry, and Ig
gene rearrangement analysis is achieveable in CLL with
fludarabine and is predictive of the response duration.
o 1992by The American Society of Hematology.
T
received prior cytotoxic therapy. The median number of prior
therapies was two. The distribution according to Rai stage was Rai
0,6%; Rai I, 19%; Rai II,24%; Rai III,24%; and Rai IV, 27%. The
median follow-upwas 36 months.
Diagnostic criteria. All patients fulfilled the National Cancer
Institute-Sponsored Working Group diagnostic criteria for CLL.9
Pretreatment evaluation included a medical history, physical examination, complete blood cell count, differential analysis, platelet
count, chemical survey, bone marrow examination,Ig quantitation,
lymphocyte immunophenotype evaluation, and Ig gene rearrangement analysis.
Treatment. Fludarabine (30 mg/m2intravenously [IV] daily for
5 days) plus prednisone (30 mg/m2 orally daily for 5 days) were
administered approximatelyevery 4weeks for a total of six courses.
Patients were thoroughly reevaluated after every three and six
courses of therapy. The majority of patients received six courses of
treatment. There was no statistically significant difference in the
number of treatments in any of the subgroups.
HE NATURAL history of chronic lymphocytic leukemia (CLL) has been carefully described by a number
of investigators.14 Over the years, the often indolent nature
of this disease has been emphasized. This feature, combined with the frequent advanced age of CLL patients, the
palliative effect of available therapy, and concerns of
further immunosuppression from treatment, initially resulted in a relatively low level of interest in the development
of new and innovative treatment strategies. Recently, with
the identification of several active agents and
interest in clinical research in CLL has been revitalized.
Consequently, both the National Cancer Institute-Sponsored Working Group and The International Workshop on
CLL have recommended objective guidelines for patient
eligibility and response criteria to facilitate clinical trial
c o m p a r i s ~ n . ~Both
J ~ working groups stress the need to
pursue minimal residual disease assessment and note the
persistence of residual lymphoid marrow nodules as the
only clinical evidence of disease in some responding patients.
Herein, we summarize the results of residual disease
evaluation in CLL by clinical parameters, bone marrow
histopathology, flow cytometry using dual-parameter immunofluorescence staining, and Ig gene rearrangement analysis in a large number of patients treated with fludarabine
plus prednisone. The clinical relevance and implications of
these findings are addressed.
PATIENTS AND METHODS
Patients. One hundred sixty-one patients with progressive or
symptomaticCLL entered into a clinical trial between August 1988
and December 1989. The median age was 62 years (range 34 to 82
years). One hundred sixteen (73%) were male and 123 (77%) had
Blood, VOI 80, NO 1 (July 1). 1992: pp 29-36
From the Departments of Hematology, Laboratory Medicine, and
Pathology, The University of Teras, M.D. Anderson Cancer Center,
Houston.
Submitted October 11, 1991; accepted March 3, 1992.
Presented in part at the combined American Society of Hematology
and International Society of Hematology Meeting, Boston, M4,
December 1990.
Address reprint requests to L.E. Robertson, MD, Department of
Hematology, The University of Taus, M.D. Anderson Cancer Center,
1515 Holcombe Blvd, Box 61, Houston, TX 77030.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I 8 U.S.C. section I734 solely to
indicate this fact.
8 1992 by The American Society of Hematology.
0006-4971 l92l8001-0020$3.00/0
29
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ROBERTSON ET AL
30
Response criteria. The sites considered for clinical response
included the peripheral blood, bone marrow, lymph nodes, spleen,
and liver. The criteria for response are shown in Table 1. These
criteria are similar to the National Cancer Institute-Sponsored
Working Group recommendations9 with only minor differences:
bone marrow examinations were performed at the end of the last
treatment; a 50% decrease in marrow lymphoid infiltration was
required for a partial response (PR); and a normocellular marrow
for age was not required (however, adequate values for hemoglobin
concentration, absolute neutrophil count, and platelet count as
defined in Table 1were required). In addition, complete response
(CR) patients were stratified based on the presence or absence of
residual marrow lymphoid findings into CR and nodular CR (nCR)
groups. We defined clinical relapse as an increase in the absolute
lymphocyte count above lO,OOO/~L,more than 50% lymphocytes
on marrow differential analysis, more than a 50% increase in the
sum of the products of at least two lymph nodes, the appearance of
new lymph nodes, more than 50% increase in the liver/spleen span
below the costal margin, the new appearance of palpable hepatosplenomegaly, or the development of an aggressive lymphoma.
Pathologic review. All responding patients underwent posterior
iliac crest marrow biopsies at both treatment initiation and
response evaluation. Marrow biopsy specimens from CR patients
(both CR and nCR) were evaluated by two pathologists (J.J.B. and
W.C.P.) for the presence of residual lymphoid findings. Two
patients were excluded from analysis after pathologic review and
reclassified as leukemic-phase small-cleaved cell lymphoma based
on paratrabecular involvement and lack of CD5 expression on B
lymphocytes. Marrow biopsies were evaluated for cellularity, percentage lymphocytes, and pattern of bone marrow infiltration. Five
different patterns were recognized: (1) normal, no evidence of
lymphoproliferation in the bone marrow; (2) nodular pattern,
nodules of small mature lymphocytes that lack clear germinal
centers; (3) interstitial pattern, replacement of normal hematopoietic tissue by small mature lymphocytes infiltrating between fat
without distortion of marrow architecture; (4) mixed pattern, both
nodular and interstitial involvement; and (5) diffuse pattern,
eradication of marrow architecture by small mature lymphocytes.
Zmmunophenotyping. Immunophenotyping was performed on
peripheral blood and bone marrow samples collected in EDTA by
flow cytometry using a simultaneous dual-color staining technique,
before treatment and after three courses and after six courses of
fludarabine. The following combinations of phycoerythrin (PE)conjugated and fluorescein isothiocyanate (F1TC)-conjugated
monoclonal antibodies (MoAbs) were used: IgG2-PE/lgGl-FITC
(control), CD14-PE/CD45-FITC, HLA-DR-PE/CDZFITC, CD4PE/CD&FITC, CD20-PE/CDS-FITC, CDS-PE/IgM+D-FITC,
CD~-PE/K-FITC,and CDS-PE/A-FITC. Ten microliters of each
MoAb was added to 0.5 x lo6 to 1.0 x lo6 cells and incubated at
2°C to 8°C for 15 minutes in the dark. The red blood cells in the
sample were lysed using ammonium chloride for 10 minutes
followed by a washing step. (For staining SIgM + SIgD, K and A,
the last two steps are reversed in sequence). The cells were
resuspended and fixed with 1% paraformaldehyde. The analysis
was performed by FACScan (Becton-Dickinson, San Jose, CA)
using a Consort 30 program. Residual disease was determined by
coexpression of CD5 on B lymphocytes in conjunction with
monoclonality of surface light-chain expression on CD5 positive B
cells (Fig 1). The presence of more than 10% of the total
lymphocyte population coexpressing CD20 and CD5 with monotypic light-chain expression was considered positive for residual
disease. A K:X or A:K ratio exceeding 3:l was considered as
monotypic light-chain expression (Fig 1).
Ig gene rearrangement detection. Ig gene rearrangement detection was performed on marrow aspirates from all patients before
therapy. Approximately 20% of the responding patients had repeat
gene rearrangement study after six courses of fludarabine. DNA
from the marrow aspirate cell pellets was purified by standard
phenol/chloroform extractions and ethanol precipitation after
proteinase K digestion. Ten-microgram samples of DNA were
digested individually to completion with the following restriction
endonucleases: EcoRI, HindIII, and BamHI. The samples were
electrophoresed on a Probe Tech I (Oncor, Gaithersburg, MD)
using a 0.7% agarose gel and transferred to a nylon filter (Oncor),
in accordance with the manufacturer's instructions. Hybridization
studies were performed with the following oligolabeled DNA
probes: JH, Ig heavy-chain J region (Oncor); JK, Ig K light chain J
region (Oncor); CA, Ig A light chain C region (courtesy of Dr P.
Leder, Harvard Medical School, by licensing arrangement); and
TCP, T-cell receptor P-chain gene constant regions C l and C2
(Oncor). After hybridization the Southern blots were exposed to
Kodak X-OMAT AR x-ray film (Eastman Kodak, Rochester, NY)
for 3 days at -70°C. For cases lacking sufficient DNA to set up the
required enzyme digests for all the lineage probes simultaneously,
previously probed membranes were stripped of the radioactive
probe and rehybridized with additional probes in accordance with
the manufacturer's instructions.
Statistical analysis. For comparison of distinct variables between two groups, either the two-sample t-test or the x2 test was
used to determine statistical significance. Actuarial survival and
time to progression were measured from the date of treatment
initiation. The endpoint for time to progression was clinical
relapse, as previously defined under response. Survival and time of
progression were estimated by the method of Kaplan and Meier."
The log-rank test was used to assess statistical significance between
subgroups.12
RESULTS
All 159 patients were assessed for response. The response rates after six courses of fludarabine plus prednisone are shown in Table 2. A CR or an nCR was obtained
in 12% and 30% of patients, respectively. The PR rate was
18% for an overall response rate of 60%. The overall
response rate was 81% in early Rai stage (0 to 11) patients
versus 36% in advanced Rai stage (I11 and IV) patients.
The failure rate was 40% with 48 of 64 patients having
refractory disease and 16 of 64 dying while on study. Five
Table 1. Criteria for Response in CLL
Response
Blood*
Marrow
CRt
14.000 LymphocyteslpL
<30% Lymphocytes on aspi-
PR
2 50% Decrease lymho-
ration differential
250% Decrease in infiltrate*
cytes/pL
LiverISpleen
Nodes
Impalpable
No pathologic nodes
250% Decrease in span below costal margin
250% Decrease
~
*CR and PR patients must have a neutrophil count 1,5OO/pL, platelet count 2 100,000 pL, and an untransfused hemoglobin 2 11.O g/dL.
t C R Patients are further divided into CR and nCR groups based on the presence or absence of residual marrow lymphoid findings.
*Infiltrate, lymphocytes x marrow cellularity.
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31
MINIMAL RESIDUAL DISEASE IN CLL
-1
U
0
100
101
-
102
103
104
10’
100
CD5 Fluoroscein
-
102
103
56.2%1
1
-
Lambda Fluoroscein
Kappa Fluoroscein
P
0
r
86.0%
- .. ... ..
30.3%
0.2%
l-
0.6%
1
0.3%
0.6%
0
?:?..
’
Fig 1. (A) Two-color immunofluorescencecontour plot of bone marrow in an nCR case demonstratingresidualdisease. Approximately 30% of
lymphocytes coexpress CD5 and CD20 with monotypic light chain. ( 8 )Two-color immunofluorescencecontour plot of bone marrow in a CR case
demonstratingno residual disease. Less than 1% of lymphocytes coexpress CD5 and CD20 without monotypic light chain.
patients achieved a response compatible with CR (one),
nCR (three), or PR (one), but failed to have normalization
of their peripheral counts. The overall median survival time
is 120 weeks. An improved CR and nCR rate was noted
for patients receiving fludarabine as frontline therapy
(P < .002).
In the 66 patients meeting clinical criteria for a complete
response, residual marrow lymphoid findings were identified in 47 patients. The bone marrow infiltration patterns
observed included: nodular, 27 patients; interstitial, 14
patients, and mixed, 6 patients. By definition, all CR
patients (both CR and nCR) had less than 30% lymphocytes on marrow aspirate differential analysis. Minimal
variation was observed on the extent of involvement between groups on aspiration differential evaluation. However, evaluation of the extent of lymphoid involvement on
Table 2. Responseto Fludarabine
Response
CR
Nodular
PR
Fail
Tota I
Patients ( O h )
Prior Therapy
Patients (%)
No Prior
Therapy
Patients ( O h )
19 (12)
47 (30)
29 (18)
64 (40)
10 (8)
34 (28)
21 (17)
58 (47)
9 (25)
13 (36)
8 (22)
6 (17)
marrow biopsy evaluation showed significant differences.
Comparison of the lymphocyte marrow biopsy infiltrate
(cellularity x percentage lymphocytes/lOO) showed patients with any nodular involvement to have significantly
higher marrow lymphocyte infiltrate (mean 5 SEM,
12.1 -r- 12.0) than patients with pure interstitial findings
(mean ? SEM, 7.85 f 7.6) (P < .01).
To investigate if biopsy assessment of the marrow infiltration pattern would be of clinical utility in determining time
of progression, the CR and nCR groups were compared. A
statistical difference was observed with 87% of CR patients
versus 55% of nCR patients progression-free at 2 years
(P < .03) (Fig 2). Not shown is the time to progression in
the PR patients, which was remarkably similar to that
observed in patients with residual lymphoid marrow findings.
Further separation of the nCR group into patients with
any residual lymphoid nodules, or those with solely interstitial involvement, also showed differences in time to progression. Patients with any nodular involvement had a longer
progression-free interval than those with purely interstitial
involvement at 2 years (63%v 39%, P < .02).
Analysis of overall survival showed no significant difference in complete responders subgrouped by the marrow
findings (CR v nCR) (P= not significant [NS]). However,
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ROBERTSON ET AL
32
1.oo
0.80
5- 0.60
2n 0.40
0.20
l
0
26
I
1
,
1
,
52
78
WEEKS
1
104
t
l
130
Fig 2. Time to progression for patients with CR and nCR. (A) CR:
total 19, fail 5; (B) nCR: total 47, fail 28; P < .03.
three of the four CR patients who died had no clinical
evidence of relapse. In contrast, in the 10 deaths in the nCR
group, six of eight evaluable patients had clinical evidence
of disease recurrence. Patients who obtained a PR or for
whom fludarabine failed had much shorter survival (Fig 3).
Surface immunophenotyping by flow cytometry using
dual-color staining on the peripheral blood and bone
marrow was performed on all of the 66 complete responders and showed no residual disease in 89% of patients
obtaining a CR. In the nCR and PR groups, the percentage
of patients demonstrating no residual disease on twoparameter flow cytometry was less (51% and 19%, respectively). Further subgrouping of the nCR patients showed
that residual disease detection was more common in patients with interstitial involvement versus those with purely
nodular involvement (74% v 42%). In the PR patients
demonstrating no residual disease on two-color flow cytometry, persistent disease was clinically outside the peripheral
blood and bone marrow compartments and limited to
residual lymphadenopathy. In the failure group, residual
disease was detected in all patients tested.
Comparison of flow cytometry taken simultaneously from
the peripheral blood and bone marrow showed a high
degree of concordance. However, in approximately 5% of
cases residual disease was detected in the marrow aspirate
and not detected in the peripheral blood.
Achievement of no residual disease status by dualparameter immunofluorescence staining in complete responders (CR and nCR) was a highly significant predictor
of time to progression. For complete responders having no
residual disease on flow cytometry the 2-year progressionfree survival rate was 84% versus 39% in patient having
residual disease detected by two-color flow cytometry
(P < .001) (Fig4).
In the 12 relapses that occurred in patients demonstrating no residual disease on dual-parameter flow after six
courses of fludarabine, eight had evidence of CLL in the
blood detected by two-color flow cytometry at relapse.
Detection of disease by flow cytometry preceded clinical
1.oo
7
0.80
E+
B0
-4
5E
0.60
K
K
E
g
0
E
0.40
0.20
I
0
0.40
I
I
0.20
I
,
,
,
,
,
,
.
,
.
,
26
52
78
WEEKS
104
130
Fig 3. Overall survival for patients with CR, nCR, PR, and no
response. (A) CR: total 19, four failures; (B) nCR: total 47,lO failures;
IC) PR: total 29,15 failures; (D) no response: total 64,53 failures; P <
.001.
0.60
t
l
0
n
l
26
*
t
52
-
78
l
~
104
~
130
WEEKS
Fig 4. Time to failure in CR patients with residual flow detected
disease and without residual flow detected disease. (A) No residual
disease: total 39,12 failures; (B) residual disease: total 27,21 failures;
P < .001.
*
l
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MINIMAL RESIDUAL DISEASE IN CLL
33
relapse in six patients from 3 to 14 months. In four patients,
no interim analysis was obtained. In the three patients with
negative flow cytometry at the time of relapse, the relapse
was caused by isolated progressive lymphadenopathy including two patients who developed a more aggressivehistology
(Richter’s Syndrome).
Pretreatment and posttreatment Ig gene rearrangement
detection using JH, JK, and Ch probes was performed in a
number of responding patients. One or more rearranged Ig
heavy- and light-chain genes were observed before treatment initiation. In five of seven patients obtaining a CR, a
return to the germline configurationwas noted. In contrast,
no rearranged bands were detected in only two of eight of
the nCR patients. In all of the PR patients and nonresponders undergoing repeat testing, rearranged bands were
demonstrated. There was no discordance between residual
disease detection by dual-parameter flow cytometry and
gene rearrangement study. None of the seven patients who
demonstrated no detectable Ig gene rearrangement have
yet relapsed.
The pretreatment characteristics and posttreatment findings of the 19 patients obtaining CR are shown in Table 3.
These patients ranged in age from 35 to 76 years (median,
64 years). Eleven patients (58%) were men, and 11 (58%)
had received prior treatment. The initial Rai stage ranged
from 0 to IV (median, 11), and diffuse marrow lymphocyte
infiltration at presentation was observed in 58%. After
treatment, no residual marrow lymphoid findings were
observed. Seventeen of 19 (89%) had no detectable residual disease by two-parameter flow cytometry, and five of
seven (71%) had no evidence of clonal Ig gene rearrangement. Time to last follow-up was 51 to 182 weeks. Five
patients have relapsed and four died. Four of the relapse
patients remain alive. Causes of death included small cell
lung cancer in one patient and infection in three patients
(listeriosis, pneumocystis, and bacterial pneumonia).
DISCUSSION
Comparison of treatments for CLL in many earlier
studies is difficult because of marked variations in the
criteria for response. Earlier clinical trials failed to use
uniform response criteria, and bone marrow evaluation was
often not included in the response assessment. Because of
an increase in the number of clinical trials in CLL, the
National Cancer Institute-Sponsored Working Group and
the International Workshop on CLL have published criteria for eligibility and r e s p o n ~ e . ~Both
J ~ working groups note
the frequent occurrence of residual lymphoid marrow
nodules. The differentiation of benign lymphocyticnodules
from small lymphocytic lymphoma/CLL can be difficult,
even on repeat biopsy.13 Because of this difficulty in
establishing a malignant nature, an equivocal diagnosis
must be frequently made. As a result, they are generally
considered compatible with a CR.
In contrast to the National Cancer Institute and the
International Workshop guidelines, we further stratified
complete responders based on the presence or absence of
residual lymphoid findings. The response rates in this group
of patients treated with fludarabine plus prednisone is
similar to the response rates reported with fludarabine as a
single a g e ~ ~ tParalleling
.~.~
the overall improvement in
response, patients in the no prior therapy group were more
likely to demonstrate no residual marrow findings than the
prior therapy group. Comparison of the CR and nCR
groups showed significantlyimproved progression-free survival in patients having no residual lymphoid marrow
findings (P < .03). Among the nCR patients, the nodular
pattern group had a longer progression-free interval than
Table 3. CR Patient Characteristics
Pretreatment
Patient
No.
1
27
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Posttreatment
YrslSex
Prior
Therapy
PB
Lymphocytes
( X 109IL)
Pattern
741M
6OlF
67lM
7OlF
76lF
35/F
68lM
69lF
57lM
70lM
48lM
62lF
69lM
53lM
48lM
64lM
69lF
46lM
4OlF
No
No
No
No
Yes
Yes
Yes
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
7.6
52.2
42.0
14.3
67.5
152.4
10.4
28.8
61.7
45.3
17.0
87.1
103.2
3.8
108.5
46.0
192.8
270.5
272.0
Diffuse
Interstitial
Interstitial
Interstitial
Diffuse
Diffuse
Mixed
Diffuse
Diffuse
Interstitial
Interstitial
Diffuse
Diffuse
Diffuse
Interstitial
Interstitial
Diffuse
Diffuse
Diffuse
Abbreviations: PE, peripheral blood; EM, bone marrow.
Time (wks) to:
BM
Rai
Stage
IV
I
0
0
II
0
I
0
II
II
II
II
II
IV
IV
111
111
II
IV
Residual
Flow
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
Yes
No
No
Residual
DNA
No
No
No
No
No
No
No
-
-
Relapse
Death
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
Yes
104
0
111
94
0
0
-
0
Yes
110
61
0
0
-
-
-
0
0
1
0
1
0
Last
Follow-up
(wk)
128
101
136
82
51
182
162
142
122
122
135
140
140
134
119
66
89
162
77
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
34
the group with only interstitial involvement. This is similar
to previous studies in lymphoma where a nodular growth
pattern was a favorable predictive feature.14J5Patients with
nodularity also demonstrated less residual disease detectable by flow cytometry than patients with interstitial involvement. Whether this is because some of these nodules are
truly benign lymphocytic nodules or because of greater
difficulty in aspirating these nodules remains speculative.
The bone marrow infiltration pattern at diagnosis is a
well-recognized prognostic feature based on several independent investigations.16-z0Based on multivariant analysis
of 329 patients, Rozman et aImreport that the bone marrow
histologic pattern is the best single prognostic parameter in
CLL. Our data suggest that the marrow findings posttherapy are also important in assessing the response to therapy
and remission duration.
To further define CR, dual-parameter immunofluorescence flow cytometry was used to detect minimal residual
disease. B-cell CLL cells typically display low-density surface Ig. Traditionally, clonality in B-cell CLL has been
defined by the expression of a single light chain, either K or
In addition, CLL cells usually express B-cell-associated
antigens such as CD19 and CD20. A highly characteristic
feature of B-cell CLL lymphocytes is the expression of the
nominal T-cell-associated antigen, CD5,22,23which has also
been identified on a subpopulation of normal B cells in fetal
cord blood, spleen, and lymph nodes and at the periphery of
the germinal center in adult lymph nodes.” This characteristic coexpression of CD5 with B-cell-associated antigens in
CLL and monoclonality of surface light-chain expression
can be easily and rapidly identified by dual-parameter flow
cytometry. Using dual-parameter flow cytometry, a state of
no residual disease was detected in a greater number of
complete responders having no residual lymphoid findings,
suggesting that the residual marrow lymphocytes in some of
the patients represent CLL cells. Comparison of time to
progression was consistent with this, as over 60% of the
nCR patients relapsed in 2 years compared with only
approximately 15% of the CR patients (P < .03).
The concordance rate between residual disease detected
by flow cytometry simultaneously taken from the blood and
marrow compartments was high. However, in 5% of patients having differing results residual disease was detected
in the marrow and not the blood, suggesting the marrow
may be a more sensitive site for residual disease detection.
In patients in the flow cytometry negative group who had
a clinical relapse, a proportion of cases were predicted
earlier by repeat flow cytometry. In three patients there was
no evidence of residual disease detectable by two-color flow
cytometry in the peripheral blood and bone marrow at the
time of relapse. All three of these patients had recurrent
disease limited to lymphadenopathy, and Richter’s syndrome was diagnosed in two of these patients. This supports the need for further close clinical follow-up in
patients, despite continued negative posttreatment flow
cytometry.
Ig gene rearrangement studies using JH, JK, and Ch
probes was performed in a proportion of CR patients.
Despite the small number tested, there was a significant
difference in the obtainment of germline rearrangement
ROBERTSON ET AL
pattern in CR patients compared to patients with residual
marrow abnormalities. This finding, in conjunction with the
dual-parameter flow cytometry findings, lends further support to the notion that these residual lymphocytes may
represent CLL. It is noteworthy that the dual-parameter
surface immunophenotype results were concordant with
the molecular studies and none of the patients who demonstrated no residual disease on gene rearrangement study
have yet relapsed.
Brugiatelli et aIz studied CLL patients obtaining a
complete clinical remission after treatment with chlorambucil. Single-parameter surface lymphocyte analysis by flow
cytometry showed 16 of 28 patients had normalization of
circulating B cells as measured by mouse rosette-forming
cells, SmIg, and CD24 labeling. All but six patients had an
abnormal K-A ratio and only one of nine patients studied
lacked evidence of clonal Ig gene rearrangement. A significant improvement in the remission duration was observed
in patients having no detectable residual disease. Using
dual-parameter flow cytometry, Vuillier et a P evaluated 22
CLL patients with a complete clinical remission confirmed
by marrow examination. Eight patients were in a phenotypic remission. Clinical correlation with relapse pattern
was not made in this preliminary analysis. In addition to
lymphocyte surface marker analysis, Ig gene rearrangement
assessment appears to provide information. Soper et alz7
sequentially studied 12 CLL patients and found clinical
status correlated with changes in the relative proportion of
cells with germline versus rearranged Ig genes determined
by densitometric quantification. Our analysis of nine patients returning to a germline pattern also suggests that this
is also of value in determining freedom from progressive
disease.
Other possible methods to assess the completeness of
cytoreduction in CLL include cytogenetic analysis, fluorescence in situ hybridization (FISH), evaluation for residual
monoclonal idiotype expression, and the polymerase chain
reaction (PCR). Serial karyotypic analysis using an abnormal/normal metaphasez8 ratio or FISHz9 could further
define the completeness of response. However, this requires a specific chromosomal abnormality, and approximately 50% of CLL patients are d i p l ~ i d ? ~
The
, ~ ~use of
anti-idiotype detection requires knowledge of the idiotypic
determinant in each case, and the more sensitive technique
of DNA amplification by the PCR requires prior knowledge
of the unique Ig gene rearrangement. All of these methods
are more labor intensive compared with marrow histopathology assessment and dual-color flow cytometry.
The observation that response to therapy in CLL correlates with a significant benefit in survival has been made in
numerous clinical t r i a l ~ . 5 , ~Such
~-~~
observations do not
necessarily imply that the efficacy of therapy resulted in
improved survival, as responders must live long enough to
achieve a response.37Comparison of survival in two groups
that have reached the clinical landmark of CR is subject to
less bias. The striking findings of this study were the marked
increase in progression-free survival in responding patients
having no residual lymphoid marrow biopsy findings and no
residual disease detectable by dual-parameter flow cytometry. The comparison of overall survival within complete
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MINIMAL RESIDUAL DISEASE IN CLL
35
responders was not significant, perhaps because of the
present duration of follow-up. In addition to predicting the
duration of remission, the evaluation of the completeness
of cytoreduction can be used to better select patients for
bone marrow harvest and future autologous bone marrow
transplant.
In the past, the attainment of CR was rare in CLL. With
fludarabine, this goal is achievable. Our results have implications for the further definition‘ of CR in future clinical
trials in CLL. Morphologic assessment of the bone marrow
biopsy and flow cytometry using dual-parameter staining
easily and rapidly measure the quality of CR. These
additional endpoints can be used for interim comparative
analyses of clinical trials and appear to be of clinical value
based on significant improval in durability of remission and
a trend toward increased survival in patients with minimal
residual disease. Further study is required to demonstrate if
more complete eradication of the malignant clone will be
beneficial to overall survival and quality of life in CLL.
ACKNOWLEDGMENT
The authors thank Dean Anthony and Mary Chandler for expert
secretarial assistance. We also thank Sherry Pierce and Susan
Lerner for their expert assistance in data management.
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1992 80: 29-36
Response assessment in chronic lymphocytic leukemia after
fludarabine plus prednisone: clinical, pathologic, immunophenotypic,
and molecular analysis [see comments]
LE Robertson, YO Huh, JJ Butler, WC Pugh, C Hirsch-Ginsberg, S Stass, H Kantarjian and MJ
Keating
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