Hematopathology / TYPICAL AND ATYPICAL CHRONIC LYMPHOCYTIC LEUKEMIA
Typical and Atypical Chronic Lymphocytic Leukemia Differ
Clinically and Immunophenotypically
John L. Frater, MD,1 Karen F. McCarron, MD,1 Jeffrey P. Hammel, MS,2 Joel L. Shapiro, MD,1
Michael L. Miller, DO,1 Raymond R. Tubbs, DO,1 James Pettay, MT(ASCP),1
and Eric D. Hsi, MD1
Key Words: Atypical chronic lymphocytic leukemia; Chronic lymphocytic leukemia; Flow cytometry; Polymerase chain reaction;
Fluorescence in situ hybridization
Abstract
We compared the features of 17 cases of atypical
chronic lymphocytic leukemia (aCLL) with those of a
clinical control group of 24 cases of CLL. Quantitative
flow cytometric data, available for 12 cases, were
compared with an immunophenotypic control group of 58
cases using a relative fluorescence index for CD5, CD23,
CD79b, and surface immunoglobulin light chain (sIg).
Compared with the clinical control group, patients
with aCLL had a higher mean WBC count and a lower
platelet count. Patients with aCLL had a significantly
higher probability of disease progression. Compared
with an immunophenotypic control group of 58 CLL
cases, 12 cases of aCLL demonstrated significantly
higher expression of CD23. There was no significant
difference in expression of sIg, CD79b, or CD5 between
the groups. CD38 expression was noted in only 1 (9%)
of 11 tested cases; 2 (18%) of 11 cases had trisomy 12.
aCLL can be distinguished from typical CLL
morphologically, clinically, and immunophenotypically.
Atypical morphologic features in CLL seem to be a
marker of aggressive clinical behavior.
© American Society of Clinical Pathologists
Chronic lymphocytic leukemia (CLL) constitutes the
majority of cases of chronic lymphoid leukemias and typically
has an indolent clinical course. However, a subset of patients
with CLL experience a more aggressive clinical course and
worse prognosis. One morphologic feature that has been
shown to be prognostically important is an increased population of peripheral blood prolymphocytes, and the FrenchAmerican-British (FAB) classification of leukemias reflects
this.1,2 However, certain patients without an increased prolymphocyte population also have an accelerated, unremitting clinical course.3,4 Several researchers have attempted to link a
variety of variables to the poor prognosis in this group, such as
abnormal lymphocyte morphologic features and karyotype
(eg, trisomy 12) of the neoplastic population.3,5-14
Two subtypes of mixed cell type CLL are proposed in
the FAB system. The first, which is synonymous with chronic
lymphocytic leukemia/prolymphocytic leukemia (CLL/PLL),
is defined as having a dimorphic population of small lymphocytes and prolymphocytes in the peripheral blood, the
prolymphocyte population constituting more than 10% and
fewer than 55% of the circulating lymphocytes. The second
subtype is defined as a “spectrum of small to large lymphocytes” with fewer than 10% prolymphocytes in the peripheral
blood ❚Table 1❚ .1 Recent studies have established a link
between CLL/PLL and karyotype abnormalities involving
chromosome 12.4,5,15,16
Both subtypes fail to take into account the patients
whose peripheral blood lymphocytes resemble circulating
lymphoma cells, with nuclear folds and deep clefts. Early
studies that correlated abnormal cell morphologic features
with poor prognosis were done before the advent of multiparameter flow cytometry and may have included cases of
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❚Table 1❚
Definitions of Types of B-Cell CLL and PLL*
Type
Prolymphocytes (%)
CLL
CLL, mixed cell type
aCLL
CLL/PLL
PLL
10 or less
10 or less
10 or less
>10 and <55
55 or more
Mixture of Small
Clefted and Folded Atypical
and Large Lymphocytes
Lymphocytes (%)
No
Yes
No
No
No
<10
<10
10 or more
—
—
Usual Immunophenotype
CD5+, CD19/20+, CD23+, weak sIg
As for CLL
As for CLL
As for CLL
CD5–/+, CD19/20+, strong sIg
aCLL, atypical CLL; CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia; sIg, surface immunoglobulin.
* Adapted from Bennett et al1 with addition of current definition of aCLL.
mantle cell lymphoma, follicle center lymphoma, and other
low-grade lymphoproliferative disorders with similar cell
morphologic features.11-13 A more recent study10 established
the existence of cases of immunophenotypically proven
CLL with the same nuclear irregularities. A poor prognosis
was shown in this series to be independent of chromosome
12 abnormalities. 10 There is a relative lack of studies
comparing immunophenotypic data in cases of typical and
atypical CLL (aCLL) as defined by at least 10% circulating
lymphocytes having prominent nuclear clefts and folds. We
compared the morphologic, clinical, and immunophenotypic
differences between these 2 groups to determine whether
patients with CLL with a cytologically atypical peripheral
blood lymphocyte population have predictable clinical and
immunophenotypic differences from patients with conventional CLL.
❚Image 1❚ Morphologically atypical chronic lymphocytic
leukemia (aCLL). Lymphocytes from cases 4 (upper right and
lower right) and 16 (upper left and lower left), showing small
to medium-sized cells with prominent nuclear clefts (WrightGiemsa, original magnification ×400).
656
Am J Clin Pathol 2001;116:655-664
Materials and Methods
Patient Selection and Control Groups
During a 29-month period, two of us (J.L.F. and E.D.H.)
identified 17 cases of aCLL seen during routine clinical
service work at the Cleveland Clinic Foundation, Cleveland,
OH, based on blood smear morphologic features and an
immunophenotype consistent with CLL (ie, a CD5+, CD19+,
CD23+ monotypic B-cell population). This comprised
approximately 7% of the total CLL cases seen during this
time. A minimum of 10% atypical lymphocytes with prominent nuclear folds and clefts in the earliest available peripheral blood smear (4 months or less after original diagnosis in
11 patients; 7 months after original diagnosis in patient 4; 24
months after original diagnosis in patient 9; 30 months after
original diagnosis in patient 12; 36 months after original diagnosis in patient 7; and more than 36 months after original
diagnosis in patients 14 and 15) was necessary for inclusion
in the study group. We defined a nuclear cleft as extending at
least 50% through the diameter of the lymphocyte nucleus
❚Image 1❚. Earliest available CBC count data were recorded
for the patients with aCLL. They were derived from the time
of diagnosis for patients 1 through 6 and 10 through 17. The
data for patients 7, 8, and 9 were from 36 months, 144
months, and 18 months after diagnosis, respectively. The
CBC and manual differential data used for inclusion are from
the same specimens for patients 1 through 3, 5, 6, 8, 10, 11,
13, 16, and 17. Rai stage was from the original date of diagnosis and was determined from the clinical data or (when not
complete) from direct reference to Rai stage in the patient
record or discussion with the clinician. Clinical variables of
interest included clinical stage at diagnosis and time from
original diagnosis to clinical stage progression or death.
The clinical control group has been described previously.17 It consisted of 24 patients fulfilling FAB criteria for
the diagnosis of CLL (CD5+, CD19+, CD23+ monotypic
surface immunoglobulin light chain [sIg] and more than
5,000/µL [5 × 109/L] peripheral blood lymphocytes with
round nuclear contours and condensed chromatin).1
© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
A separate control group of 58 patients with typical
CLL, previously described, was used when comparing the
results of quantitative flow cytometric immunophenotyping
(FCI) for the patients with aCLL.18 These patients fulfilled
the same inclusion criteria as the clinical control group, ie,
more than 5,000/µL (5 × 109/L) peripheral blood lymphocytes with morphologic features of typical CLL and the
immunophenotype as noted in the previous paragraph.
approved alpha satellite (centromere region, 12 p11.1-q11.1)
CEP 12 SpectrumOrange DNA probe kit (Vysis, Downers
Grove, IL) according to the manufacturer’s specifications. 4',6diamidino-2-phenylindole II counterstain (Vysis) then was
applied to each slide. Signal enumeration was performed as
recommended by the manufacturer. Per protocol, 300 lymphoid
cells were counted on each patient’s slide; more than 5%
trisomic cells was considered a positive result.
Flow Cytometric Immunophenotyping
Four-color FCI was performed for all patients (n = 17)
and for both control groups of typical CLL cases. Antibodies
for CD5, CD19, CD23, kappa, and lambda were used for all
cases.18 The atypical CLL cases and phenotypic control
group cases diagnosed after September 1998 also were
analyzed for FMC7 (not evaluated in all cases) and CD79b.18
A quantitative estimate of fluorescence was performed
on 12 of the 17 aCLL cases and the 58 CLL cases from the
flow cytometry control group. The flow cytometer was calibrated daily to standard calibration beads (Becton Dickinson,
San Jose, CA). In addition, for the phycoerythrin (PE)
channel, an additional calibrator was used to achieve identical
performance of each of the instruments (Spherotech, Libertyville, IL). Antibodies to CD5 fluorescein isothiocyanate
(FITC), CD23 PE, CD79b PE, FMC7 FITC, kappa FITC, and
lambda PE were used. All antibodies were manufactured by
Becton Dickinson except CD79b (Immunotech, Marseilles,
France). A quantitative value was assigned to the expression
of each marker using the relative fluorescence index (RI,
mean channel number of the positive population divided by
autofluorescence for the appropriate fluorochrome). A
CD45+/CD19+ gating strategy was used to ensure that only B
cells were analyzed. In addition, FCI for CD38 PE (Becton
Dickinson) was performed in the same manner for 9 patients
with aCLL. CD38 positivity was defined as at least 30% positive B cells compared with the autofluorescence control.
Cyclin D1
In 9 aCLL cases, paraffin-embedded cell block preparations were made of Ficoll-Hypaque (Amersham Pharmacia
Biotech, Uppsala, Sweden)–separated lymphocytes. After
centrifugation of the samples, 3 or 4 drops of normal human
plasma was added to each specimen; 3 or 4 drops of thrombin
then was added to each, and 10 mL of 10% buffered formalin
was added to the resultant clots. They then were processed and
embedded in paraffin. Cyclin D1 immunoperoxidase stains
were performed on 5-µm-thick sections cut from the cell
blocks on an automated instrument (clone DCS6, Ventana,
Tucson, AZ). Positive control immunostaining consisted of a
formalin-fixed known positive mantle cell lymphoma.
Cytogenetics
Conventional cytogenetics was performed in the cytogenetics laboratory of the Cleveland Clinic Foundation for 6
aCLL cases using an approved method.19 Briefly, blood
lymphocytes were stimulated with 12-O-tetradecanoyl
phorbol-13-acetate in supplemented RPMI media (20% calf
serum, 10% penicillin/streptomycin, 0.3% L-glutamine)
(Sigma Aldrich, Indianapolis, IN) for 3 to 5 days. Cell
pellets prepared from this solution then were washed with
Carnoy fixative. Slides then were prepared with trypsin
Giemsa (G) banding of the chromosomes.20
Statistical Analysis
Statistical analysis of a number of clinical variables of
interest was performed to describe and compare the aCLL
group and the clinical control group. These variables included
WBC count, hemoglobin concentration, and platelet count at
diagnosis and subsequent changes in these values. The
Wilcoxon rank sum test or t test was used to compare the
aCLL and control groups with respect to the means of quantitative data. Log transformation and analysis of covariance
was used to adjust for stage with respect to the CBC data. The
Rai stage at diagnosis was noted for all patients, and any
interval change in stage was noted.23 An increase in clinical
stage was noted as “progression.” A Kaplan-Meier curve and
log-rank test were used to compare the aCLL and control
groups. A Cox proportional hazards model was used to adjust
Fluorescence In Situ Hybridization
In 9 cases, fluorescence in situ hybridization (FISH) was
performed using a US Food and Drug Administration–
© American Society of Clinical Pathologists
Polymerase Chain Reaction for t(11;14)
Evaluation for t(11;14)(q13;q32) was performed on
DNA extracted from peripheral blood lymphocytes from 9
aCLL cases by polymerase chain reaction using primers for
the major translocation cluster. The t(11;14)(q13;q32) primer
sequence was as follows: mantle cell lymphoma–1, 5'-GAT
GGG CTT CTC TCA CCT ACT A-3'; and the Jh consensus
primer was as follows: Jha, 5'-ACC TGA GGA GAC GGT
GAC C-3'.21 Beta globin served as the amplification control,
and a t(11;14)-positive cell line was used as positive control.
The beta globin primer sequences were 5'-CCA CTT CAT
CCA CGT TCA CC-3' (upstream) and 5'-GAA GAG CCA
AGG ACA GGT AC-3' (downstream).22
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❚Table 2❚
Morphologic and Laboratory Features of Atypical Chronic Lymphocytic Leukemia Cases*
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
WBC
(/µL)
Hemoglobin
(g/dL)
Platelet Count
(× 103/µL)
PMNs
(%)
Lymphocytes
(%)
Monocytes
(%)
33,400
208,000
15,320
42,940
403,000
35,800
35,360
31,800
40,780
27,490
21,600
24,160
48,300
21,830
19,000
66,600
26,800
13.6
10.1
14.7
NA
NA
NA
8.8
12.0
10.9
11.8
12.0
NA
8.9
14.7
NA
12.9
14.7
147
188
123
NA
NA
NA
32
NA
199
113
264
NA
114
148
NA
182
110
36
1
10
3
2
14
0
19
18
8
20
0
3
18
3
5
32
60
97
87
95
96
86
98
76
78
86
67
100
93
80
94
91
61
1
0
1
1
0
0
0
2
3
2
6
0
0
1
0
1
5
Prolympho- Eosinocytes
phils
(%)
(%)
3
2
2
1
2
0
2
3
1
4
4
0
4
1
1
3
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
1
0
2
Promyelo- Atypical Lymphocytes
cytes
(per 100 Lymphocytes)
(%)
(%)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
16
22
24
26
10
10
13
10
14
14
15
11
14
14
10
19
21
NA, value not measured or not available.
* WBC, hemoglobin, and platelet and differential counts are from the earliest available CBC counts and blood smears, respectively. Conversion factors and units of measure for
Système International units are as follows: WBC, multiply by 0.001, × 109/L; hemoglobin, 10.0, g/L; platelet count, 10.0, × 109/L; lymphocytes, monocytes, and eosinophils,
0.01 (proportion of 1.00).
❚Table 3❚
Clinical Features of Patients With Atypical Chronic Lymphocytic Leukemia
Rai Stage
Case No./
Sex/Age (y)
At Diagnosis
At Most Recent
Follow-up
Follow-up From
Original Diagnosis (mo)
1/M/61
2/F/70
1
3
4
3
15
4
3/M/68
4/F/47
5/F/92
6/M/52
7/M/78
8/M/62
9/F/72
10/F/74
11/M/75
12/M/73
13/F/76
14/M/72
15/F/59
16/F/90
17/M/68
1
0
2
0
0
0
0
0
3
0
3
0
0
3
0
4
1
2
0
4
1
0
4
3
3
3
2
2
3
0
23
7
0
13
40
148
24
11
4
41
4
156
180
27
4
for the differences between the patients and the 2 groups with
respect to age, sex, and stage at diagnosis.
Results
Clinical Findings
Nine men and 8 women were identified with aCLL. The
clinical and laboratory features are shown in ❚Table 2❚ and
❚Table 3❚. The mean and median ages of patients at diagnosis
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Treatment
Fludarabine, prednisone
Chlorambucil, prednisone, fludarabine,
interferon alfa
None
None
None
None
Prednisone
None
None
Chlorambucil, fludarabine
None
None
None
None
Fludarabine
None
None
were 69.9 years and 72 years at diagnosis. The distribution
of Rai stages at diagnosis was as follows: stage 0, 10 cases;
stage 1, 2 cases; stage 2, 1 case; and stage 3, 4 cases. All
patients had peripheral blood lymphocytosis with a monotypic CD5+, CD19+, CD23+ B-cell population.
A mean of 15.47% (range, 10%-26%) of the circulating lymphocytes in the earliest available blood smear
had the atypical feature of deeply clefted and folded
lymphocyte nuclei. A mean of 1.94% (range, 0%-4%) of
the circulating WBCs in the earliest available blood smear
© American Society of Clinical Pathologists
Estimated Progression-Free Probability
Hematopathology / ORIGINAL ARTICLE
1.0
0.8
Typical CLL
0.6
0.4
0.2
aCLL
0.0
0
10
20
30
40
50
60
Months
❚Figure 1❚ Kaplan-Meier time-to-event analysis. Comparing
the atypical chronic lymphocytic leukemia (aCLL) group with
the CLL clinical control group, the former has a statistically
greater probability of clinical progression compared with the
latter (Cox proportional hazards model; P = .028, adjusted for
age, sex, and stage at diagnosis).
were prolymphocytes. Compared with the clinical control
group, patients with aCLL had a higher mean WBC count and
lower mean platelet count at diagnosis. In an unadjusted
analysis, this was found to be statistically significant (P = .002
and P = .014, respectively). After adjustment for stage, these
differences in presenting CBC indices were still statistically
significant (P = .001 and P = .013, respectively). In 3 patients,
the earliest available CBC data were from a variable period
after original diagnosis (36 months for patient 7; 144 months
for patient 8; and 18 months for patient 9). In a separate
analysis of the CBC data not including these 3 patients, similar
results were obtained, with a statistically significant difference
between the 2 groups with regard to WBC count and platelet
count at diagnosis (P = .002 and P = .043, respectively). Five
patients (29%) received nonanthracycline pharmacotherapy
including fludarabine (n = 4), prednisone (n = 3), chlorambucil (n = 2), and interferon alfa (n = 1). The remaining 12
patients (71%) received no specific therapy.
Mean follow-up times were 20 months for the clinical
control group and 41 months for the aCLL group. With regard
to clinical progression, the disease in 11 patients progressed
from the Rai stage at diagnosis; 9 of these were from the aCLL
group, and 2 were from the clinical control group. Since followup times varied, a Kaplan-Meier time-to-event analysis was
most appropriate for comparing the groups ❚Figure 1❚. With the
log-rank test, the clinical control group was less likely to show
clinical progression (P = .001). The same conclusion was made
even after adjusting for the effects of age, sex, and stage at diagnosis (P = .028) using a Cox proportional hazards model. An
attempt was made to adjust for absolute lymphocyte count and
WBC count in addition to the aforementioned variables.
However, several patients from the aCLL group (patients 4, 7, 9,
12, 14, and 15) could not be included in this analysis since their
CBC and differential data were not from the same period. This
resulted in a loss of statistical power. However, a statistical trend
still was observed (P = .07). Five patients died of causes not
directly related to CLL (patients 2, 10, 11, 13, and 14).
Flow Cytometric Immunophenotyping
By definition, all cases from both groups were CD5+,
CD19+, CD23+, sIg+. Quantitative FCI data were available for
12 cases of aCLL and all 58 immunophenotypic control cases.
There were significant differences between the groups when
analyzed quantitatively ❚Table 4❚ and ❚Table 5❚. The aCLL
group had significantly brighter expression of CD23 (165.53 vs
91.71; P = .04) ❚Figure 2❚. Although the aCLL group had an
average brighter expression of CD5 (17.91 vs 14.83), CD79b
(14.21 vs 7.30), and sIg (42.92 vs 12.01), these differences
were not significantly significant at the .05 level (t test).
In addition, given that recent data suggest a poor prognosis of CD38+ CLL,24 9 aCLL cases were evaluated for the
❚Table 4❚
Results of Quantitative Flow Cytometric Immunophenotyping and Assessment of CD38*
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
15
CD5
CD23
FMC7
CD79b
Surface Immunoglobulin
27.21
10.79
10.08
4.61
7.62
24.42
25.16
49.91
10.38
23.52
2.38
18.87
ND
37.47
467.51
177.63
80.17
75.65
149.17
241.97
265.52
142.21
221.83
12.30
116.07
ND
3.38
4.59
37.10
8.77
10.91
3.67
4.30
5.82
27.41
3.80
26.56
5.48
ND
1.34
14.16
12.92
18.65
6.67
5.54
8.14
9.75
45.94
5.82
37.4
4.15
ND
2.47
13.86
123.63
214.43
24.32
1.99
6.48
7.78
21.91
3.43
92.66
2.19
ND
CD38
ND
Negative
ND
ND
ND
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
ND, not done.
* All values are expressed as mean fluorescence intensity index.
© American Society of Clinical Pathologists
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❚Table 5❚
Quantitative Flow Cytometric Data Comparison for Atypical Chronic Lymphocytic Leukemia and Control Groups*
Study group (n = 12)†
Mean
Median
Range
Control group
Mean
Median
Range
t Test
CD5
CD23
CD79b
Surface Immunoglobulin
17.91
14.83
2.38-49.91
165.53
145.69
12.3-467.51
14.21
8.95
1.34-45.94
42.92
10.82
1.99-214.43
14.83 (n = 50)
13.80
2.41-31.12
NS
91.71 (n = 50)
73.95
2.66-311.35
P = .04
7.30 (n = 58)
7.14
1.14-51.46
NS
12.01 (n = 55)
6.29
0.77-139.64
NS
NS, not statistically significant.
* All values are expressed as mean fluorescence intensity (log scale).
† Data are given for 12 of the 17 patients for whom quantitative data were complete.
presence or absence of this antigen by flow cytometry. CD38
expression was noted in only 1 (11%) of 9 aCLL cases tested,
with high intensity expression in the majority of cells.
One patient with trisomy 12 by conventional cytogenetics
also had positive results by FISH (Image 2). FISH was
not performed for the other trisomy 12 case identified by
conventional cytogenetics.
BCL-1/Cyclin D1
In all 9 aCLL cases analyzed, PCR for t(11;14) was
absent using major translocation cluster primers. In these
same 9 patients, the results of cyclin D1 immunoperoxidase
stains performed on cell block specimens also were negative.
In addition, results for 2 other aCLL cases in which biopsy
tissue was available were negative using immunoperoxidase
stains. The first was performed on a lymph node biopsy
specimen demonstrating involvement by CLL, the second on
an involved bone marrow biopsy specimen.
A
B
150
150
Counts
Counts
Karyotyping/FISH for Trisomy 12
Given the association between atypical morphologic
features and trisomy 12, we studied cases with available
cells for trisomy 12. A total of 2 (18%) of 11 cases
studied by FISH, cytogenetics, or both had trisomy 12.
Conventional cytogenetic analysis was performed on 6
patients with aCLL ❚Table 6❚. Two patients had trisomy 12
by this method ❚Image 2❚ . In addition, FISH was
performed on 9 aCLL cases, including 4 of the patients
having conventional karyotype analysis. No discrepant
results were noted in the cases analyzed by both methods.
0
0
100
101
102
CD23 PE
103
104
100
101
102
103
104
CD23 PE
❚Figure 2❚ CD23 expression in typical (A) and atypical (B) chronic lymphocytic leukemia (aCLL). Cases of aCLL had an average
brighter expression of CD23 compared with the control group, as exemplified in these 2 cases. Shaded curve indicates CD23
expression; dashed curve indicates autofluorescence control. PE, phycoerythrin.
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© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
❚Table 6❚
Cytogenetic Findings and Assessment of BCL-1 and Cyclin D1
Case No.
1
2
3
6
7
8
9
10
11
12
15
Cytogenetics
46,XY
ND
47,XY,+12
46,XY
ND
ND
47,XX,+12
45X,–X,inv(9)c/46XX*
ND
ND
46,XX
Chromosome 12 FISH
ND
Normal
ND
Normal
Normal
Normal
Trisomy 12
Normal
Normal
Normal
Normal
BCL-1
ND
Negative
ND
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Cyclin D1
Negative (lymph node)
Negative
Negative (bone marrow)
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
FISH, fluorescence in situ hybridization; ND, not done.
* inv(9) is a familial benign polymorphism present in approximately 1% of the general population.
Discussion
We report the results of a study examining the clinical
and immunophenotypic differences separating CLL from
morphologically atypical CLL. Compared with a clinical
control group composed of patients with typical CLL,
patients with aCLL had a higher likelihood of clinical
progression. Patients with aCLL had a greater average
expression of CD23 compared with the immunophenotypic
control group of morphologically typical CLL cases. For
these reasons, aCLL, as we have defined it, seems to have
morphologic, immunophenotypic, and clinical features that
distinguish it from typical CLL.
The significance of atypical morphologic features in
CLL has been a source of controversy for many years. The
earliest series, reported before the routine use of FCI, may
have contained cases that would today be diagnosed as
other lymphoproliferative disorders.11,12 This may account
in part for the adverse clinical outcome attributed to cases
having morphologically atypical lymphocytes. After the
widespread use of FCI, researchers initially concluded that
cases of CLL having 5% lymphocytes with nuclear clefts
showed no difference in prognosis compared with typical
CLL.6,25 This viewpoint was contradicted by later work.
Que et al26 noted that patients meeting FAB criteria for
CLL/PLL had a greater likelihood of having atypical
lymphocyte morphologic features and trisomy 12 compared
with patients with typical CLL. Matutes et al9 and Oscier et
al10 noted an increased incidence of trisomy 12 and adverse
clinical course in patients with CLL with more than 15%
cleaved cells or lymphoplasmacytoid cells. Criel et al,27
reporting a series of “atypical” CLL cases, noted a number
of unusual immunophenotypic findings. FMC7 positivity
was found in 11 of 21, CD5 negativity in 8 of 21, and
bright sIg expression in 6 of 21 cases. Atypical was defined
as a lack of any morphologic or immunophenotypic feature
© American Society of Clinical Pathologists
of typical CLL. Therefore, cases of morphologically typical
CLL with abnormal immunophenotype may have been
included in this series.27
This last example underscores a problem that is encountered commonly when reviewing the aCLL literature: Welldefined and accepted criteria for the identification of aCLL
do not exist, making it difficult to compare major series. Our
suggestion that aCLL be defined as a chronic lymphoid
leukemia with an immunophenotype of typical CLL (CD5+,
CD19+, CD23+, monotypic sIg) and a minimum of 10%
lymphocytes with clefted and folded nuclei in the peripheral
blood is one attempt at offering uniform criteria for the identification of aCLL cases. In our experience, nearly every case
❚Image 2❚ (Case 9) Chromosome 12 fluorescence in situ
hybridization. Using a centromeric probe, 3 copies of
chromosome 12 are seen in the nucleus of this lymphocyte
(SpectrumOrange DNA probe with 4’,6-diamidino-2phenylindole counterstain, original magnification ×1,000).
Am J Clin Pathol 2001;116:655-664
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of CLL contains rare cells with atypical nuclear features. In
most cases, these cells seem to constitute no more than 2% to
3% of the circulating lymphocytes. Most observers would
dismiss a small percentage of atypical lymphocytes in a
background lymphocytosis composed of typical CLL cells.
We chose 10% as a minimum percentage of atypical
lymphocytes for aCLL because of the ease of identification
of such a population at low magnification (×100). In our
experience, this percentage of clefted or irregular lymphocytes would suggest a circulating non-Hodgkin lymphoma as
a differential diagnostic consideration.
We defined the atypical cells as deeply clefted lymphocytes in a background of otherwise normal CLL cells. These
cells had condensed chromatin, were small to medium sized,
were slightly larger on average than typical CLL lymphocytes, and had at least 1 prominent nuclear cleft. Cells with
minor nuclear irregularities and indentations were not
included. Most of these cells lacked prominent nucleoli,
although a single small nucleolus sometimes was detected.
We did not include cases with increased circulating prolymphocytes meeting criteria for mixed type CLL or cases with
increased numbers of so-called prolymphocytoid cells, as has
been done in other series.3,5,27 Because an increased population of prolymphocytes is known to be associated with an
adverse clinical course, we wanted to define the impact of
cells with clefted nuclei outside the context of the presence
of an increased population of prolymphocytes.2,28 Patients
with atypical peripheral blood morphologic features of this
type represent a distinct minority of all CLL cases. In our
experience, aCLL cases represent approximately 7% of all
CLL cases.
A number of our findings differ somewhat from those
found elsewhere in the aCLL literature. Trisomy 12, associated by other researchers with abnormal cell morphologic
features,5,9,27,29 was noted by us in only 2 of 11 aCLL cases
examined. This suggests that trisomy 12, although associated
in these 2 cases with the aforementioned features, cannot
account for these findings in all patients. Moreover, a recent
study that compared trisomy 12 and abnormalities of the
TP53 tumor suppressor gene on chromosome 17p13.1 found
that adverse prognosis correlated with the latter but not with
the former.30 No abnormalities of chromosome 17 were
noted in the cases in this series examined using conventional
cytogenetics.
Besides the trisomy 12 and 17p13 abnormalities, other
genetic aberrations such as those involving chromosome
13q14 and 11q have been described in CLL.31,32 We noted
no abnormalities besides trisomy 12 in the cases studied by
karyotyping. One patient was noted to have inv(9) in all
analyzed lymphocytes, but this is a defined benign variant
present in approximately 1% of the general population.33
Most recently, certain abnormalities, notably deletion of 17p
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Am J Clin Pathol 2001;116:655-664
and 11q, but not trisomy 12, have been associated with a
poor prognosis in CLL. 34 It has been noted that CD38
expression correlates with immunoglobulin heavy chain variable gene mutation status and poor response to multiagent
chemotherapy.24,35 CD38 expression was noted in only 1 of 9
patients tested. This suggests that the poor clinical course of
our aCLL patients cannot be ascribed to CD38 expression.
With regard to the expression of abnormal markers in
CLL and aCLL, we did not examine the cases for the expression of CD11c. Expression of this marker in CLL has been
the subject of some controversy and contention, as levels of
expression have differed markedly in various series.36,37
Marotta et al38 found relatively weak expression of this
marker in CLL compared with cases of hairy cell leukemia
and prolymphocytic leukemia. In addition, expression may
be related to stage of disease in CLL, with greater expression
of CD11c in early disease states.38 In one major series,
CD11c expression did not correlate with either disease
progression or treatment-free survival.39
Patients with aCLL had a greater probability of progression to a higher stage compared with the clinical control
group. After an average of 41 months of follow-up, 5 of 17
patients were dead, none due to disease. This is a phenomenon common in the study of chronic lymphoproliferative
disorders: patients tend to be middle-aged to elderly at onset
and many die of unrelated causes. A longer follow-up time is
necessary to determine any difference in overall survival. We
recognize the fact that this is a retrospective series of patients
and that there may be some heterogeneity, particularly since
2 of our patients were identified long after the original diagnosis. Even when these 2 patients were excluded from the
progression analysis, a statistically significant difference was
present. Whether all these patients had similar morphologic
features at the time of diagnosis and throughout the disease
course is unknown. It will be necessary to observe our
patients over time to determine whether these morphologic
features persist and progress. Since our clinical progression
data were calculated from the date of original diagnosis, it
seems that atypical morphologic features may be a marker
for progression regardless of when the features are noted.
Although none of the patients were treated with anthracycline-containing protocols, we also recognize that treatment
was not uniform. However, these patients may benefit from
alternative therapies. Prospective studies are required to fully
validate these findings.
We studied the immunophenotypic, cytogenetic,
morphologic, and clinical differences between CLL and
aCLL. We have suggested a relatively straightforward means
to distinguish CLL from aCLL and aCLL from other chronic
lymphoproliferative disorders. A continuing problem is the
lack of a consistent definition for the entity that clearly separates it from other diseases. By applying the definition we
© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
have provided, which requires only standard FCI and
morphologic review, further studies can be conducted to
confirm these findings.
From the 1Division of Pathology and Laboratory Medicine and
2Department of Biostatistics, Cleveland Clinic Foundation,
Cleveland, OH.
Presented, in part, at the annual meeting of the United States
and Canadian Academy of Pathology, New Orleans, LA, March
28, 2000.
Address reprint requests to Dr Hsi: Division of Pathology
and Laboratory Medicine, Cleveland Clinic Foundation, 9500
Euclid Ave, Cleveland, OH 44106.
Acknowledgments: We thank the personnel of the molecular
pathology and flow cytometry laboratories at the Cleveland Clinic
Foundation for technical assistance.
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