Hematopathology / PRECURSOR B LYMPHOBLASTIC LEUKEMIA
Precursor B Lymphoblastic Leukemia With Surface Light
Chain Immunoglobulin Restriction
A Report of 15 Patients
Rina Kansal, MD,1 George Deeb, MD,1 Maurice Barcos, MD, PhD,2 Meir Wetzler, MD,3
Martin L. Brecher, MD,4 AnneMarie W. Block, PhD,5 and Carleton C. Stewart, PhD6
Key Words: Precursor B cell; Acute lymphoblastic leukemia; Surface immunoglobulin–positive acute leukemia; Flow cytometry; WHO
classification; Immunophenotyping
DOI: 10.1309/WTXCQ5NRACVXTYBY
Abstract
We describe 15 patients (9 children) with precursor
B-cell (pB) acute lymphoblastic leukemia (ALL) with
surface immunoglobulin (sIg) light chain restriction
revealed by flow cytometric immunophenotyping (FCI).
The same sIg+ immunophenotype was present at
diagnosis and in 3 relapses in 1 patient. In 15 patients,
blasts were CD19+CD10+ (bright coexpression) in 14,
CD34+ in 12, surface κ+ in 12, surface λ+ in 3; in 8 of
8, terminal deoxyribonucleotidyl transferase (TdT)+;
and in 4, surface IgD+ in 2 and surface IgM+ in 1. The
3 CD34– cases included 1 TdT+ case, 1 with
t(1;19)(q23;p13), and 1 infant with 70% marrow blasts.
One adult had CD10–CD19+CD20–CD22+CD34+
TdT+sIg+ blasts with t(2;11)(p21;q23). Blasts were L1
or L2 in all cases (French-American-British
classification). Karyotypic analysis in 12 of 12
analyzable cases was negative for 8q24 (myc)
translocation. Karyotypic abnormalities, confirmed by
fluorescence in situ hybridization in 6 cases, included
hyperdiploidy, t(1;19)(q23;p13), t(12;21)(p13;q22),
t(9;22)(q34;q11), t(2;11)(p21;q23), and trisomy 12.
The sIg light chain restriction in pB ALL might be
present in neoplasms arising from the early,
intermediate, and late stages of precursor B-cell
maturation; sIg light chain restriction revealed by FCI
does not necessarily indicate a mature B-cell
phenotype, further emphasizing the importance of a
multidisciplinary approach to diagnosing B-lymphoid
neoplasms.
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Neoplasms of the B-lymphoid cell may have a precursor
B-cell phenotype or a mature B-cell phenotype, as described
in the World Health Organization (WHO) classification of
hematopoietic malignant neoplasms.1 The latter category
includes Burkitt leukemia/lymphoma and other neoplasms of
the mature B-cell, which may have a leukemic manifestation.
In the diagnostic evaluation of these B-cell neoplasms, flow
cytometric immunophenotypic (FCI) analysis has a critical
role in the differentiation of a precursor B-cell phenotype
from a mature B-cell phenotype.2 While a mature B-cell
phenotype shows the presence of surface immunoglobulins
by FCI analysis, this expression typically is absent in a
precursor B-cell phenotype. Precursor B-cell (pB) acute
lymphoblastic leukemia (ALL) is a neoplasm of B
lymphoblasts that characteristically are negative for surface
light chain immunoglobulins by FCI analysis and express
markers related to the degree of B-cell differentiation.1-3
Cases of pB ALL with the expression of immunoglobulins
include those described as “transitional pre-B ALL,” which
express cytoplasmic and surface IgM but no surface light
chain immunoglobulins.4
The expression of surface light chain immunoglobulins
in pB ALL seems to be rare in the pediatric age group.5 In
adults, we found 7 published cases of ALL with surface light
chain immunoglobulin restriction6,7 that would be classified
as pB ALL by the WHO classification. Previous authors
have suggested that the lymphoblasts in these pB ALL cases
might represent a B-cell stage intermediate between the transitional pre–B-cell stage and the mature B cell.6,7 We present
findings from 15 patients with pB ALL with surface light
chain immunoglobulin restriction, diagnosed by a multidisciplinary approach in a single institution, which demonstrate
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
for the first time that surface light chain immunoglobulin
restriction in precursor B lymphoblasts is not confined to
any particular stage of B-lymphoblast differentiation and,
instead, may be seen in a heterogeneous group of pB ALLs
showing different degrees of differentiation of pB
lymphoblasts. To the best of our knowledge, this is the
largest reported group of cases with this unusual
immunophenotypic expression in pB ALL.
Materials and Methods
This single-institution retrospective study was designed
at Roswell Park Cancer Institute (RPCI; Buffalo, NY)
following an index case of pB ALL with surface light chain
immunoglobulin restriction, which was observed in the clinical practice of one of us (R.K.) at Buffalo General Hospital,
Buffalo, NY. The flow cytometry database at RPCI was
searched for the period January 1995 to December 2001 for
all cases of “acute lymphoblastic leukemia” and for all “Blineage malignancies” with surface immunoglobulin (sIg)
expression. The pathology database also was searched to
identify cases of pB ALL.
At least 198 cases of ALL were identified; however, the
cases retrieved by the specific key word searches represented
only a subset of the total number of cases of ALL analyzed
by flow cytometry at RPCI, precluding an estimate of the
incidence of sIg+ pB ALL in our cases. The immunophenotypic and relevant pathologic data were reviewed, and 19
cases of pB ALL were identified in 15 patients, showing sIg
light chain–restricted neoplastic cells by FCI analysis. All
cases were classified according to the WHO criteria.1 Cases
of Burkitt lymphoma/leukemia were excluded. The clinical,
morphologic, cytogenetic, and flow cytometric immunophenotypic findings were reviewed in all 19 cases. Clinical
information, including age, sex, sites of involvement by
disease, leukocyte counts at diagnosis, and treatment and
clinical follow-up data were recorded by review of medical
records.
Flow Cytometry
Multiparameter flow cytometry was performed in all
cases in the Laboratory of Flow Cytometry, RPCI, as previously described.8,9 All cases were analyzed by 4-color (15
cases) or 3-color flow cytometry (1995-1997; 4 cases in 3
patients) using a FACScan Flow Cytometer (Becton Dickinson, San Jose, CA).
An acute leukemia panel of antibodies was used in all
cases. At RPCI, this panel routinely includes surface κ and λ,
HLA-DR, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b,
CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22,
CD23, CD25, CD32, CD33, CD34, CD38, CD41, CD45,
CD56, CD57, CD64, CD71, and CD79b. Surface IgD and
IgM were analyzed in 4 cases using 3-color FCI analysis.
The 4-color fluorochrome combinations included fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridinium
chlorophyll protein complex (PerCP) or PE-cyanin-5 (PECY5
or Tricolor (TC, Caltag, Burlingame, CA)/allophycocyanin
(APC). The 3-color fluorochrome combinations included
FITC/PE/PerCP or PECY5 or TC. The corresponding antibody
combinations included CD3/CD14/PerCP-HLA-DR/CD45,
CD3/CD4/TC-CD8/CD45, CD7/CD13/TC-CD2/CD19,
CD5/λ/PECY5-CD19/κ, CD20/CD11c/TC-CD22/CD25,
CD5/CD19/PECY5-CD10/CD34, CD11b/CD13/PECY5CD33/CD34, CD15/CD56/PECY5-CD19/CD34, and
IgD/IgM/PECY5-CD19. The surface κ and λ antibodies used
were APC-κ (clone TB28-2, BD Biosciences, San Jose, CA)
and PE-λ (clone 1-155-2, BD Biosciences). All other antibodies were obtained from the following sources: Caltag (CD2,
CD8, CD14, CD15, CD16, CD19, CD22, CD23, CD32,
CD41, CD57, CD71, CD79b, IgD, and IgM), BD Biosciences
(CD3, CD4, CD5, CD11c, CD13, CD14, CD20, CD25, CD34,
CD45, CD64, and HLA-DR), and Immunotech-Coulter,
Hialeah, FL (CD7, CD10, CD11b, CD19, CD33, CD38,
CD56, and CD71).
The samples analyzed included 18 bone marrow aspirates and 1 peripheral blood sample. Cell viability was determined by using ethidium monoazide fluorescence, as previously described.10 All specimens exhibited more than 98%
viable cells after processing. All data were obtained in list
mode and displayed as 2-parameter dot plots using WinList
multiparameter analysis software (Verity Software House,
Topsham, ME). The antibody panel that best resolved
leukemia cells was chosen to identify the cell population of
interest, and the data were reanalyzed (“back gated”) to
produce bivariate displays in the forward vs the side scatter
plot. New regions were drawn around the cells of interest in
this display (“leukemia gate”), and these scatter regions then
were used to analyze all other panels. Cells stained with
isotype-matched antibody-fluorochrome combinations and
biologically negative control cells within each tube were used
as negative controls. The cell clusters of interest were called
positive for a surface antigen if they showed a distinct shift in
fluorescence intensity in comparison with the negative
controls. The intensity of fluorescence was reported as dim if
the mean fluorescence intensity was within the second logarithmic decade (the first logarithmic decade being negative).
Morphologic Examination and Terminal Deoxyribonucleotidyl Transferase Immunohistochemical Analysis
The corresponding peripheral blood smears, bone
marrow aspirate smears (Wright-Giemsa–stained and cytochemically stained), and H&E-stained bone marrow aspirate
clot sections and core biopsy sections were reviewed. The
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French-American-British (FAB) morphologic type of the
lymphoblasts11 and bone marrow blast percentages were
recorded. In patients with lymph node and bone marrow
involvement, more than 25% bone marrow blasts were
required for the diagnosis of pB ALL, as opposed to fewer
than 25% marrow blasts and fewer than 10% circulating
blasts that would be classified as lymphoblastic lymphoma
according to the Pediatric Oncology Group criteria.12 Retrospective assessment for nuclear terminal deoxyribonucleotidyl transferase (TdT) enzyme could not be performed
by flow cytometry because additional cryopreserved cell
samples were not available for study. Therefore, an immunohistochemical stain for TdT (TdT, DAKO, Carpinteria, CA)
was performed in all cases with available paraffin-embedded
sections (cases 7, 10-15) or unstained air-dried marrow aspirate smears, using the DAKO automated staining system.13
The aspirate smear slides were fixed in cold methanol (4°C,
15 minutes) before antigen retrieval. In all cases, a
microwave oven (100°C, 14 minutes, Target Retrieval Solution [DAKO], pH 8.9-9.9) was used for antigen retrieval.
Conventional Cytogenetics and Fluorescence In Situ
Hybridization
Conventional cytogenetic analyses were performed on
trypsin–Wright stain banded bone marrow.14 Cells were
examined from 24- or 48-hour unstimulated cultures or from
methotrexate-synchronized unstimulated cells cultured for 24
hours. At least 20 cells per case were examined whenever
possible. Chromosomal abnormalities were described
according to the International System for Human Cytogenetic
Nomenclature (1995).15 Fluorescence in situ hybridization
(FISH) analyses were performed using commercially available (Vysis, Downers Grove, IL) α satellite centromeric and
locus-specific rearrangement probes in 6 cases. At least 500
interphase nuclei were examined for FISH analysis.
Results
Clinical and Morphologic Features for 15 Patients
The clinical and morphologic features for all patients
are given in ❚Table 1❚. The 15 patients included 9 children
(cases 1-9, Table 1) with an age range of 11 months to 13
years (median, 5 years) and 6 adults (cases 10-15, Table 1)
with an age range of 20 to 73 years (median, 60 years). All
15 patients were given a diagnosis of pB ALL by a multidisciplinary approach on referral to RPCI, with the unusual
finding of surface light chain immunoglobulin–restricted
lymphoblasts by FCI analysis. The white blood cell counts at
diagnosis ranged from 840 to 169,100/µL (0.8-169.1 ×
109/L; median, 4,200/µL [4.2 × 109/L]).
The corresponding Wright-Giemsa–stained bone marrow
aspirate smears were available for review in all cases. By
FAB morphologic type, the blasts were L1 or L2 in all cases
❚Image 1❚ . Bone marrow blast percentages ranged from
38.5% to 97.4% (median, 91%) in the smear preparations.
Enzyme cytochemical stains on smear preparations were
❚Table 1❚
Clinical and Pathologic Features of 15 Patients With Precursor B Lymphoblastic Leukemia
Sites
Case No./Sex/
Age (y)*
LN
Spleen/
Liver†
1/F/3
2/M/3
3/M/3
4/F/5
5/M/7
6/M/9
7/M/12
8/M/11 mo
9/F/13
10/M/20
11/M/34
12/M/56
13/F/62
14/F/67
15/F/73
No
Yes
No
No
Yes
No
Yes
Yes
Yes
No
Yes
No
No
No
No
No/2-3
2/2
No/No
No/No
No/No
No/No
No/4
Tip/No
No/2-3
No/No
No/No
No/No
No/No
No/No
No/No
Blasts
Mediastinum/
CNS
No/No
No/No
No/No
No/No
No/No
No/No
No/No
No/Yes
No/No
No/No
No/No
No/No
No/No
No/No
No/No
Outcome
WBC
Count (/µL‡)
BM (%)
FAB
Type
1,400
59,100
4,200
4,300
2,800
2,300
169,100
22,900
13,200
2,600
1,900
10,980
7,300
3,200
840
61.5
95.8
96.4
96.2
59.6
97.0
97.4
71.5
91.4
40.2
97.0
92.0
63.2
59.4
38.5
L1
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L2
L2
L2
L2
Time of
CR/Duration
FCI Diagnosis§
(mo)
Initial
Initial
Initial
Initial
Initial
Initial
Initial
Initial
Initial
Initial||
Initial
Initial
Relapse
Initial||
Relapse
Yes/49
Yes/17
Yes/27
Yes/21
Yes/72
Yes/25
No
Yes/57
Yes/19
Yes/4
Yes/1
Yes/22
No
No
Yes/6
Survival
Alive; NED
Alive; NED
Alive; NED
Alive; NED
Alive; NED
Alive; NED
Died; 5 mo
Alive; NED
Alive; NED
Died
Died
Alive; NED
Died
Died
Died
BM, bone marrow; CNS, central nervous system; CR, complete remission; FAB, French-American-British co-operative group11; FCI, flow cytometric immunophenotypic
analysis; LN, lymph nodes; NED, no evidence of disease.
* Unless otherwise indicated.
† Numeric values are centimeters.
‡ Values are given in conventional units; to convert to Système International units (× 109/L), multiply by 0.001.
§ The time of FCI (at initial diagnosis or relapse) that showed surface light chain immunoglobulin restriction on lymphoblasts.
|| FCI at primary diagnosis and at persistent/relapsed leukemia in these cases showed surface light chain immunoglobulin restriction on lymphoblasts.
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© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
available for review for 10 patients (cases 1-6, 8, 9, 11, 12).
By these cytochemical stains, the leukemic cells were negative for myeloperoxidase and Sudan black in smears from all
10 patients, negative for chloroacetate esterase in 9 of 9, and
showed granular periodic acid–Schiff positivity in 2% to 83%
of blasts (median, 35%) in 9 of 9. Bone marrow trephine
biopsies were performed in all adults, revealing effacement of
marrow architecture by leukemic cells in all cases.
Peripheral blood smears at the time of diagnostic bone
marrow biopsies were available for review for 1 patient
(case 12).
One adult (case 11) underwent a concurrent lymph node
biopsy at a local referring hospital at the time of diagnosis,
which was reviewed (R.K. and M.B.) and showed architectural effacement by lymphoblasts that were revealed as
CD10+TdT+ by paraffin section immunohistochemical
stains. Fresh tissue from this lymph node was not submitted
for FCI analysis. Bone marrow trephine biopsy sections in
this case, however, showed extensive bilateral effacement of
marrow architecture by lymphoblasts, consistent with pB
ALL as opposed to precursor B lymphoblastic lymphoma by
the Pediatric Oncology Group criteria.12
FCI Analysis and TdT Immunohistochemical Findings
for 15 Patients
The flow cytometric immunophenotype for all patients
is given in ❚Table 2❚. The FCI analyses for all except 2
patients were performed at the time of initial diagnosis. For 1
adult (case 12), the initial FCI analysis was done at Buffalo
General Hospital at diagnosis and was followed by repeated
diagnostic FCI analysis at RPCI. The scatter plots of both
FCI analyses for case 12 were reviewed (R.K. and C.C.S.)
and showed the same surface light chain restriction on the
lymphoblasts and additional dim surface IgM expression on
the lymphoblasts in the initial analysis.
For 1 adult (case 14), FCI analyses were done on 2
separate bone marrow aspirate samples at diagnosis and at
the time of 5 subsequent analyses, all of which were
performed at the RPCI flow cytometry laboratory and
showed persistent leukemia. The same immunophenotype
and surface light chain restriction were present in the first 5
of the 7 FCI analyses; the sixth and the seventh consecutive
analyses did not show surface light chain restriction in this
patient with persistent lymphoblastic leukemia. For 2
patients, the results of FCI analysis at the time of relapse
showed surface light chain immunoglobulin restriction.
Review of the FCI analysis results at the time of primary
diagnosis in these 2 cases did not show surface light chain
restriction on the lymphoblasts.
The blasts were CD19+ in all cases and showed bright
coexpression of CD19 and CD10 in samples from 14 of 15
patients. CD45 was dim or absent, with HLA-DR positivity
in all cases. The leukemic cells were CD34+ by FCI
analysis in 12 of 15 patients and TdT+ by immunohistochemical analysis in 8 of 8 ❚Image 2❚. The 3 CD34– cases
included 1 TdT+ case, 1 with t(1;19)(q23;p13), and 1 infant
with 71.5% bone marrow lymphoblasts. Surface CD22
expression was present on the neoplastic cells in all cases
and showed less fluorescence intensity than the residual
benign B cells in the sample. CD20 expression was dim in 2
cases and absent in 13. Dim CD5 expression was noted in
the sample from 1 infant with CD10+CD19+CD34+ blasts;
all other cases were CD5–.
The surface light chain immunoglobulin expressed was
κ in the samples from 12 of 15 patients and λ in 3 of 15, with
dim fluorescence intensity in all cases. Samples from 4
patients were analyzed for surface heavy chain immunoglobulins (cases 5, 8, 12, and 14); 2 showed IgD dim +IgM–
lymphoblasts (cases 5 and 8); case 12 showed IgM+IgD–
lymphoblasts; and the neoplastic cells in case 14 were negative for IgD and IgM. Aberrant dim expression of a myeloid
marker—CD13 or CD33—was detected on the leukemic
cells in 6 patients, with CD13+CD33+ lymphoblasts in the
samples from 3 patients, CD13+CD33– lymphoblasts in 2,
and CD13–CD33+ leukemic cells in 1. Examples of flow
cytometry plots from pediatric and adult cases of surface
light chain–positive pB ALL are shown in ❚Image 3❚, ❚Image
4❚, ❚Image 5❚, ❚Image 6❚, ❚Image 7❚, and ❚Image 8❚.
Conventional Cytogenetics and FISH Findings for 15
Patients
Cytogenetic findings are summarized in ❚Table 3❚. Karyotypic analyses were performed on samples from 12 patients,
❚Image 1❚ Several lymphoblasts are present in this bone
marrow smear from a 62-year-old woman (Wright-Giemsa,
×1,000).
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❚Table 2❚
Flow Cytometric Immunophenotypic Features for 15 Patients With Precursor B Lymphoblastic Leukemia*
Case No. TdT
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
NA
NA
NA
+
NA
NA
+
NA
NA
+
+
+
+
+
+
CD34
HLA-DR
CD45
CD19
CD10
CD22
CD20
sIg
CD13
CD33
CD5
+
+
+
+
+
+
+
–
–
–
+(d)
+
+
+
+
+
+
+(d)
+(d)
+
+
+
+
+
+(d)
+(d)
+
+(d)
+
+
+(d)
+(d)
–
+(d)
–
+(d)
+(d)
+(d)
+(d)
+(d)
+(d)
+(d)
+(d)
+(d)
+(d)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+(d)
+
+
+
+
+
+
+
+
+
–
+
+
+(d)
+
+(d)
+
+(d)
+(d)
+
+
+
+(d)
+
+(d)
+
+
+(d)
–
–
–
+(d)
–
–
–
–
–
–
–
–
–
–
λ(d)
κ(d)
κ(d)
κ(d)
κ(d)
κ(d)
κ(d)
κ(d)
κ(d)
λ(d)
κ(d)
λ(d)
κ(d)
κ(d)
κ(d)
–
–
–
+(d)
–
+(d)
+(d)
–
–
–
–
+(d)
–
+(d)
–
–
–
+(d)
+(d)
–
+(d)
+(d)
–
–
–
–
–
–
–
–
+(d)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
NA, not available; sIg, surface immunoglobulin; TdT, nuclear terminal deoxyribonucleotidyl transferase (by immunohistochemical stains); +, positive; (d), dim; –, negative.
* The samples for flow cytometric immunophenotypic analysis were bone marrow aspirates in all cases except case 12, for which a peripheral blood sample was used. Samples
were diagnostic in all cases except 13 and 15, which were samples at relapse of sIg– precursor B-cell ALL. Flow cytometric immunophenotypic analysis was 4-color in all
cases except 5, 8, and 14, in which it was 3-color. Surface IgD and IgM were analyzed in cases 5, 8, 12, and 14; see the text.
abnormalities for 2 (cases 3 and 6) without an analyzable
karyotype. FISH studies confirmed trisomies 4 and 10 in
case 1, trisomies 4 and 21 in case 2, trisomy 12 in case 4,
and bcr/abl fusion in case 7. In case 3, FISH analysis
showed 2 clonal populations: one with trisomy 4 and the
other with trisomies 4 and 10. A cryptic t(12;21)(p13;q22)
was revealed by FISH in case 6 by using the TEL/AML1
locus-specific fusion probe.
❚Image 2❚ Terminal deoxyribonucleotidyl transferase (TdT)+
lymphoblasts in paraffin-embedded core biopsy section (TdT,
immunohistochemical stain, ×400).
but samples were not analyzable for the remaining 3 patients
(cases 3, 6, and 15) owing to inadequate mitoses or a
hypocellular specimen. The karyotypes in the 12 analyzable
cases were negative for an 8q24 (myc) rearrangement. The
dividing cells showed t(1;19)(q23;p13) in 3 patients,
t(9;22)(q34;q11) in 1 patient, t(2;11)(p21;q23) in 1 patient,
trisomy 12 in 1 patient, hyperdiploidy in 3 patients, and a
normal karyotype in 3 patients.
FISH studies were performed on the samples from 6
patients. These studies confirmed karyotypic findings for 4
patients and provided evidence of specific cytogenetic
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Multiparametric Correlation in 9 Pediatric Cases
The 9 children in our study (cases 1-9) included 6 boys
and 3 girls, with ages ranging from 11 months to 13 years
(median, 5 years). The total leukocyte counts at diagnosis
ranged from 1,400 to 169,100/µL (1.4-169.1 × 10 9 /L;
median, 4,200/µL [4.2 × 109/L]).
The diagnostic bone marrow aspirate samples from all
pediatric cases were obtained at diagnosis. The
lymphoblasts in all cases showed FAB L1 or L2 morphologic features on Wright-Giemsa–stained bone marrow
aspirate smears. Bone marrow blast percentages ranged
from 59.6% to 97.4% (median, 95.8%). Bone marrow
trephine core biopsies were not performed on any of the
children. Regrettably, marrow aspirate clots or evaluable
TdT-stained aspirate smears were available for only 2 children; immunohistochemical analysis showed TdT+
lymphoblasts in these 2 cases.
FCI analysis of the samples from all patients revealed
lymphoblasts with bright coexpression of CD19 and CD10,
with surface κ light chain restriction for all except 1 patient
with λ light chain restriction (case 1). This patient was a 3year-old boy with CD5dim+CD10+CD19+CD20dim+CD34+,
surface λ+ lymphoblasts, and this seems to be the first
© American Society for Clinical Pathology
Hematopathology / ORIGINAL ARTICLE
80
60
40
104
104
103
103
CD45 APC
100
CD45 APC
Side Scatter
120
102
102
101
101
20
0
20 40 60 80 100 120
Forward Scatter
0
104
103
CD19 PE
CD19PE
103
102
101
4
102
103
CD34 APC
102
104
101
10
κ APC
102
101
101
102
103
CD19 PC
104
101
102
103
104
Anti–HLA-DR PerCP
103
102
101
4
103
λ PE
104
101
101
10
20 40 60 80 100 120
Side Scatter
CD22 TC
104
102
103
CD10 PC
104
101
102
103
CD20 FITC
104
101
102
103
κ APC
104
104
103
103
λ PE
0
102
102
101
101
101
102
103
CD19 PC
104
❚Image 3❚ (Case 2) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 3-year-old boy with CD45dim+
HLA-DR+CD10+CD19+CD22dim+CD34+ surface κdim+ leukemic cells (arrows) at diagnosis (4-color flow cytometric
immunophenotypic analysis). Sparse residual benign B cells are present in this case (arrowheads). Note that the color assigned
to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE,
phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA).
reported pediatric case of a CD5+ pB ALL with sIg light
chain restriction revealed by FCI analysis. Owing to the
coexpression of CD5 and CD19 with surface λ, which is
more common than κ in mantle cell lymphoma, 2 the
unlikely possibility of the blastoid variant of mantle cell
lymphoma in a child was considered but rejected owing to
the unequivocal presence of CD34 in FCI analysis and a
karyotype characteristic of pB ALL without the t(11;14)
translocation, in addition to the dim expression of CD45,
CD20, and surface immunoglobulin by FCI analysis that
argue against a diagnosis of mantle cell lymphoma.
For 1 child (case 4) with CD10+CD19+CD34+HLADR+, surface κ+ blasts, trisomy 12 was revealed as the sole
karyotypic abnormality. Our pediatric cases also included
cases with a cryptic t(12;21) translocation, TEL/AML1
fusion16 (n = 1), t(9;22)(q34;q11), bcr/abl fusion17 (n = 1),
and a hyperdiploid karyotype (n = 3).18 Two children in our
study had the t(1;19)(q23;p13) translocation, which often is
associated with a late stage of precursor B-cell differentiation that shows cytoplasmic IgM expression with or without
surface IgM but no surface light chain expression.19,20 The
lymphoblasts in samples from 1 of 2 patients with the
t(1;19) showed dim surface IgD κ expression, in the absence
of surface IgM expression by FCI analysis. Dim surface IgD
without surface IgM by FCI analysis also was present in 1
infant with CD10+CD19+CD20–CD22+CD34–HLA-DR+
blasts that correspond to an intermediate stage of Blymphoblast differentiation.
The 9 children were treated according to the Pediatric
Oncology Group chemotherapy protocols. All patients
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❚Image 4❚ (Case 3) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 3-year-old boy with CD45–HLADR+CD10+CD19+CD22+CD34+ surface κdim+ leukemic cells (arrows) at diagnosis (4-color flow cytometric immunophenotypic
analysis). The fluorescence intensity of CD22 on the neoplastic cells, although brighter than the CD22 intensity observed in
case 2 (Image 3), is less than that of the admixed benign B cells, which are polytypic by surface light chain immunoglobulin
expression (arrowheads). Also shown is the dim aberrant expression of CD33 on the neoplastic cells. Note that the color
assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA).
except 1 achieved complete remission and were alive with no
evidence of disease at 17 to 72 months (median, 26 months)
following complete remission.
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Multiparametric Correlation in 6 Adult Cases
The 6 adults in our study (cases 10-15) included 3 men
and 3 women, with ages ranging from 20 to 73 years
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❚Image 5❚ (Case 6) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 9-year-old boy with CD45dim+HLADR+CD10+CD19+CD22dim+CD34+ surface κdim+ leukemic cells (arrows) at diagnosis (4-color flow cytometric
immunophenotypic analysis). Residual benign B cells (arrowheads) show brighter CD22 expression in comparison with the
leukemic cells. Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC,
fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor
(Caltag, Burlingame, CA).
(median, 59 years). The total leukocyte counts at diagnosis
ranged from 840 to 10,980/µL (0.8-11.0 × 109/L; median,
2,630/µL [2.6 × 109/L]).
The leukemic samples with surface light chain
immunoglobulin restriction were obtained at diagnosis
from 4 patients and at relapse from 2 patients. The
lymphoblasts in samples from all patients showed FAB L1
or L2 morphologic features on Wright-Giemsa–stained
marrow aspirate smears. Bone marrow blast percentages
ranged from 38.5% to 97.0% (median, 61.3%).
Immunoperoxidase stains performed on paraffin-embedded
trephine core biopsy or marrow aspirate clot sections
revealed that the leukemic cells were TdT+ in all cases. By
FCI analysis, the leukemic cells coexpressed CD10 and
CD19 in samples from all patients except 1 (case 14) with
an CD10–CD15–CD19+CD22+CD20–CD34+TdT+
immunophenotype, which corresponded to an early stage of
precursor B-cell differentiation. Surface IgM and IgD were
not detectable by FCI analysis in this case, which showed
the t(2;11)(p21;q23) translocation by karyotypic analysis.
Surface IgM was present in 1 patient (case 12) with sIg+
results but without a karyotypic abnormality. Surface heavy
chain immunoglobulins were not analyzed in the sample
from the patient (case 10) with the t(1;19)(q23;p13).
In the 2 patients with surface light chain–expressing
lymphoblasts at relapse, review of the initial FCI analysis
did not show surface light chain expression on the
leukemic cells at primary diagnosis. Two patients (cases
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101
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102
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CD10 PC
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0
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❚Image 6❚ (Case 7) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 12-year-old boy with
CD45dim+HLA-DR+CD10+CD19+CD22dim+CD34+ surface κdim+ leukemic cells (arrows) at diagnosis (4-color flow cytometric
immunophenotypic analysis). Also shown is the dim aberrant expression of the myeloid markers, CD13 and CD33, on the
neoplastic cells in this case. Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin;
FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor
(Caltag, Burlingame, CA).
10 and 14) showed surface light chain–restricted
lymphoblasts at initial diagnosis and at subsequent FCI
analyses to detect residual or relapsed leukemia. In case
14, this surface light chain immunoglobulin expression
contributed to the immunophenotypic “fingerprint” of the
leukemic cells in 3 FCI analyses performed for residual
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leukemia and was no longer detectable in the fourth and
fifth consecutive FCI analyses for persistent disease.
All adults received chemotherapy according to the
Cancer and Leukemia Group B protocols. Of the 6 patients,
4 achieved complete remission, with a duration ranging
from 41 days to 666 days; however, 5 of 6 did not survive.
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❚Image 7❚ (Case 10) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 20-year-old man with
CD45dim+HLA-DR+CD10+CD19+CD34– surface λdim+ leukemic cells (arrows) at diagnosis (4-color flow cytometric
immunophenotypic analysis). Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin;
PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex.
Discussion
Accurate diagnostic distinction between pB ALL and
mature B-cell leukemia (Burkitt leukemia) is critical for
disease management. Burkitt leukemia typically shows FAB
L3 morphologic features, has a mature B-cell phenotype
(CD34–TdT–, surface light chain+) by FCI analysis and the
c-myc gene rearrangement by cytogenetics, and manifests
clinically with bulky disease that requires more intensive
chemotherapy than standard ALL therapy, with central
nervous system prophylaxis.21 However, discrepancies in the
morphologic features and immunophenotype of Burkitt
leukemia have been described. Hammami et al22 reported 9
cases of mature B-cell ALL with FAB L1 or L2 morphologic
features. Navid et al23 reported 5 pediatric cases of Burkitt
leukemia, which originally were misinterpreted as pB ALL
owing to the absence of sIg by FCI analysis. Coexpression of
precursor cell markers—CD34 and TdT—also has been
reported in Burkitt leukemia.24-26 In all of the aforementioned
cases with an aberrant immunophenotype or morphologic
features, the critical feature that determined the course of the
disease as Burkitt leukemia was the c-myc gene translocation.
All patients in our study were given a diagnosis of pB ALL
by a multidisciplinary approach, and they represent pB ALL
cases with morphologic and cytogenetic features typical of
this entity but with unexpected surface light chain
immunoglobulin restriction revealed by FCI analysis.
Our pediatric cases included cases with a cryptic t(12;21)
translocation, TEL/AML1 fusion,16 t(9;22)(q34;q11), bcr/abl
fusion,17 and a hyperdiploid karyotype.18 These cytogenetic
abnormalities are different from those reported in the 4 cases
of sIg+ pB ALL identified in a large study of pediatric pB
ALLs.5 One of our pediatric cases also showed trisomy 12,
which reportedly is uncommon in precursor B-cell
neoplasms,27,28 although it often is associated with B-cell
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0
20 40 60 80 100 120
Forward Scatter
0
104
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CD10 PC
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Anti–HLA-DR PerCP
103
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0
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CD19 PC
104
❚Image 8❚ (Case 13) Surface immunoglobulin–positive precursor B lymphoblastic leukemia in a 62-year-old woman with
CD45dim+HLA-DR+CD10+CD19+CD22dim+CD34+ surface κdim+ leukemic cells (arrows) at relapse (4-color flow cytometric
immunophenotypic analysis). Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin;
FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor
(Caltag, Burlingame, CA).
chronic lymphocytic leukemia,29 a neoplasm of mature B
cells. Of interest, trisomy 12 was reported previously in 2
adult cases of sIg+ pB ALL. In 1 case, it was part of a
complex karyotype in a 23-year-old man with
CD9+CD10+CD24+HLA-DR+TdT+, surface IgMλ+
lymphoblasts with non–FAB L3 morphologic features.6 In
our case with trisomy 12, dim aberrant expression of CD13
and CD33 also was present; these markers were not analyzed
in the case reported by Michiels and colleagues.6 Vasef and
colleagues7 reported the second adult case of sIg+ pB ALL
with trisomy 12. The immunophenotype in their case,
however, was CD5+CD10– CD19+CD20+CD34– TdT+,
surface λ+, in contrast with our case that was
CD5–CD10+CD19+CD20–CD22+CD34+, surface κ+.
The immunologic classification of ALL of B-cell
lineage is based on the stages of maturation of a B cell, with
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pB ALL arising from the early, intermediate, and late
precursor stages of B-cell differentiation.1-3,21 In addition to
nuclear TdT and HLA-DR that are expressed by B
lymphoblasts at all precursor stages of maturation, the earlystage blasts express CD19 and cytoplasmic CD22 and are
CD10–.1,21 The intermediate-stage B lymphoblasts also
express CD10, and the late precursor stage is characterized
by the expression of cytoplasmic heavy chain immunoglobulin IgM.1,21 All 6 adult pB ALL cases with surface light
chain expression described by Vasef et al7 seemed to have an
immunophenotype consistent with the most mature precursor
B-cell stage, and it was suggested that the immunophenotype
in these sIg+ pB ALL cases was intermediate between that
of pre–B cells, which are TdT+ and cytoplasmic Ig+, and
mature B cells, which express surface heavy and light chain
immunoglobulins.6,7 Our study cohort of pB ALL included
© American Society for Clinical Pathology
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❚Table 3❚
Cytogenetic Findings for 15 Patients With Precursor B Lymphoblastic Leukemia
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Karyotype
Comments
51∼57,inc [2 cells]/46,XX [1 cell]
52,XY,+X,+4,+6,+14,+21,+21 [20 cells]
Not analyzable
47,XX,?+12,inc [2 cells]/46,XX [15 cells]
60,XY,t(1;19)(q23;p13),i(7)(q10),inc [3 cells]/46,XY [17 cells]
Not analyzable
46,XY,ider(9)(q10)t(9;22)(q34;q11),der(22)t(9;22) [19 cells]/48,XY,t(9;22)(q34;q11),
+21,+der(22)t(9;22) [1 cell]
46,XY [4 cells]
46,XX,t(1;19)(q23;p13) [15 cells] / 46,XX [5 cells]
47,XY,+5,der(19)t(1;19)(q23;p13) [19 cells]/47,idem,–3,+8 [1 cell]
45,XY,?inv(10),–20 [14 cells]/46,XY [6 cells]
46,XY [20 cells]
46,XX [20 cells]
46,XX,t(2;11)(p21;q23) [20 cells]
Not analyzable
FISH +4,+10
FISH +4,+21
FISH +4/+4,+10
FISH +12
FISH t(12;21)(p13;q22),TEL/AML1 fusion
FISH bcr/abl fusion
Hypocellular specimen
Hypocellular specimen
FISH, Fluorescence in situ hybridization.
cases in the early, intermediate, and late stages of precursor
B-cell differentiation and demonstrated, for the first time,
that surface light chain immunoglobulin restriction in pB
ALL might be present in neoplasms arising from all (early,
intermediate, and late) stages of a precursor B-cell, which
otherwise have typical morphologic and cytogenetic features
of pB ALL.
Neoplastic pB cells (lymphoblasts) show aberrant
surface marker expression and deviation from the normal
sequence of maturation that can help to differentiate them
from normal marrow progenitor B cells (hematogones),30,31
which also can coexpress TdT and CD34.32 We have not
systematically assessed the expression of surface light chain
immunoglobulins on normal hematogones in our study.
Nevertheless, the neoplastic cells in our cases provide an
additional example of deviation from the normal sequence of
B-cell maturation by showing unexpected surface light chain
immunoglobulin expression, which likely would not be
present in normal hematogones. Studies of normal Blymphocyte development32 have suggested subtle differences
between the developmental stages identified in models of
normal bone marrow compared with those deduced from Blineage leukemias.33 The expression of surface light chains
on precursor B lymphoblasts in our study further suggests
that it is not always possible to correlate leukemic B cells
with normal B-cell developmental stages. One possible
mechanism that would explain the occurrence of sIg in this
heterogeneous group of pB ALL cases is uncoupling of
proliferation and maturation in B cells, which has been
reported to occur in this group of neoplasms.34 The expression of surface IgD in 2 (50%) of 4 of our cases, instead of
surface IgM that might be present in transitional pre–B-cell
ALL,4,20 also is likely an example of aberrant surface antigen
expression in neoplastic B-lineage cells.
The acquisition of surface CD22 often is considered to
indicate a more mature precursor B-cell stage. This may lead
to the argument that the presence of surface CD22 on
neoplastic cells in all our cases suggests that these actually
are examples of a more mature or “transitional” pB ALL.
There are at least 2 reasons that do not support this view.
First, the detection of surface CD22 on B-lineage
lymphoblasts most likely is fluorochrome-dependent and has
been reported in virtually all pB ALLs in at least 3 studies.35-37
Furthermore, in the flow cytometry laboratory at RPCI, all
sIg– pB ALL cases also show surface CD22 positivity,
similar to our sIg+ pB ALL cases (unpublished observation,
C.C.S.), indicating that the surface CD22 positivity in our
cases cannot be interpreted as evidence of a more mature
stage of precursor B-cell maturation.
Second, the interpretation that these cases represent a
specific precursor stage of B-cell development is not
supported by the cytogenetic findings in our cases, which
include findings considered to be typically associated with
the early-, intermediate-, and late-stage precursor B-cell
ALL groups. Instead, it is possible that these sIg+ ALL
cases arise when a subset of leukemic cells within a typical
sIg– ALL, whether early, intermediate, or late pre–B-cell
stage, progress toward greater maturational status and eventually become the predominant clone. Such a mechanism
might also explain the 2 sIg+ cases in our series that were
detected only at relapse. Nevertheless, regardless of the
underlying mechanism, these cases show that if surface light
chain restriction is detected in a neoplastic B-cell population, it does not necessarily indicate a classification of a
mature B-cell lymphoma/leukemia. In other words, a
precursor B neoplasm cannot be ruled out solely by the
presence of surface light chain immunoglobulin restriction
on neoplastic cells by using FCI analysis.
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Information from systematic studies of pB ALL that have
assessed surface light chain immunoglobulin expression on B
lymphoblasts is limited. Behm and colleagues5 reported 4
(0.7%) of 551 pediatric pB ALL cases with weak surface IgM
and λ positivity. To the best of our knowledge, the published
reports of sIg+ pB ALL in adults include a case report6 and
retrospectively identified random cases.7 Besides, the expression of surface light chain immunoglobulins on pB cells has
not been evaluated in most recent large studies with focus on
FCI evaluation of benign and neoplastic pB cells.31,38,39
Although the incidence of sIg+ pB ALL in our cases cannot
be ascertained from our present data, this unusual expression
of sIg on precursor B lymphoblasts seems to be more frequent
in our cases than currently reported in pB ALL. Furthermore,
within a period of 8 months after we closed our study, we
identified 3 additional patients given a diagnosis of sIg+ pB
ALL at RPCI (not included in this report). However, we do not
have any further explanation for the apparently increased
occurrence of sIg+ pB ALL in our cases compared with the
cases at most other tertiary institutions.
From a diagnostic standpoint, our cases demonstrate
that the expression of surface light chain immunoglobulins
on neoplastic B cells does not necessarily indicate a mature
B-cell phenotype, further emphasizing the need for a multiparametric approach in the differential diagnosis of B-cell
neoplasms, including Burkitt leukemia vs precursor B
lymphoblastic leukemia, and leukemic mantle cell
lymphoma vs pB ALL. While the c-myc gene rearrangement
would point to Burkitt leukemia in the differential diagnosis
of pB ALL from Burkitt, the differential diagnosis of sIg+
pB ALL from a leukemic manifestation of a mature B-cell
lymphoma such as the blastoid variant of mantle cell
lymphoma would be facilitated by the absence of the progenitor cell markers CD34 and TdT in the latter40; the typically
bright expression of CD20, CD45, and surface light chain
immunoglobulin in FCI analysis in the latter; and the presence of the t(11;14) translocation by cytogenetic analysis or
cyclin D1 expression by immunohistochemical analysis in
the latter.40 Furthermore, the expression of surface light
chain immunoglobulins on B-lineage lymphoblasts might be
of particular relevance in diagnosis when a limited antibody
panel must be used for FCI analysis and indicates the need
for caution in the classification of B-cell neoplasms by FCI
analysis in such instances.
The significance of this immunophenotypic finding in
the clinical course of pB ALL is not clearly determined from
our study. Nevertheless, in cases with sIg+ lymphoblasts, the
expression of sIg light chains might contribute to the diagnostic immunophenotypic fingerprint of the neoplastic cells,
as illustrated in 2 of our cases. This immunophenotypic
finding also might appear for the first time at relapse in a
typical pB ALL, as we noted in 2 cases.
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We draw the following conclusions from our study: (1)
Surface light chain immunoglobulin restriction in pB ALL
might be more common than currently recognized. (2) When
present in pB ALL, it is not confined to a late precursor stage
of B-cell differentiation, as previously suggested. Instead, it
might be present in neoplasms arising from the early, intermediate, and late stages of a precursor B cell, which otherwise have typical morphologic and cytogenetic features of
pB ALL. (3) The significance of this finding in the clinical
course of pB ALL remains to be determined. (4) From a
diagnostic standpoint, the presence of surface light chain
restriction on neoplastic B cells by FCI analysis does not
necessarily indicate a mature B-cell phenotype, emphasizing
the need for caution in evaluating limited FCI antibody
panels and the importance of multiparametric correlation in
the immunophenotypic diagnosis and classification of Blineage neoplasms.
From the 1Department of Pathology, Buffalo General Hospital, the
State University of New York at Buffalo; Departments of
2Pathology, 3Medicine, and 4Pediatrics, the 5Clinical Cytogenetics
Laboratory, and the 6Laboratory of Flow Cytometry, Roswell Park
Cancer Institute, Buffalo, NY.
Address reprint requests to Dr Kansal: Dept of Pathology,
Medical College of Wisconsin, 9200 W Watertown Plank Rd,
Milwaukee, WI 53226.
Acknowledgments: We thank Sally Bialy for assistance with
retrieval of the flow cytometry data, Mary Beth Dell for assistance
with the flow cytometry illustrations, and Bertram Schnitzer, MD,
for reviewing the manuscript.
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2. Jennings CD, Foon KA. Recent advances in flow cytometry:
application to the diagnosis of hematologic malignancy.
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3. Foon KA, Todd RF. Immunologic classification of leukemia
and lymphoma. Blood. 1986;68:1-31.
4. Koehler M, Behm FG, Shuster J, et al. Transitional pre–B-cell
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5. Behm FG, Head DR, Pui C-H, et al. B-precursor ALL with
unexpected expression of surface immunoglobulin (sIg) mu
and lambda [abstract]. Lab Invest. 1995;72:106A.
6. Michiels JJ, Adriaansen HJ, Hagemeijer A, et al. TdT positive
B-cell acute lymphoblastic leukemia (B-ALL) without Burkitt
characteristics. Br J Haematol. 1988;68:423-426.
7. Vasef MA, Brynes RK, Murata-Collins JL, et al. Surface
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