1. Healthcare

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RAPID COMMUNICATION
Leukemic Cell Growth in SCID Mice as a Predictor of Relapse in High-Risk
B-Lineage Acute Lymphoblastic Leukemia
By Fatih M. Uckun, Harland Sather, Gregory Reaman, Jonathan Shuster, Vita Land, Michael Trigg, Roland Gunther,
Lisa Chelstrom, A. Bleyer, Paul Gaynon, and William Crist
Mice with severe combined immunodeficiency (SCID) provide a model system t o examine the in vivo homing, engraftment, and growth patterns of normal and malignant
human hematopoietic cells. The relation between leukemic
cell growth in this model and the treatment outcome in
patients from whom cells were derived has not been established. Leukemic cells from 42 children with newly diagnosed high-risk B-lineageacute lymphoblastic leukemia
were inoculated intravenously into CB.17 SCID mice. Mice
were killed at 12 weeks or when they became moribund as
a result of disseminated leukemia. All mice were necropsied
and subjected to a series of laboratory studies to assets
their burden of human leukemic cells.Twenty-three patients
whose leukemic cells caused
histopathologically detectable
leukemia in SCID mice had significantly
a
higher relapserate
did not (estimated
than the 19 patients whose leukemic cells
5-year event-free survival: 29.5% Y 94.7%; 95% confidence
intervals, 11.2% t o 50.7% v 68.1% t o 99.2%; P < .OOO1 by
log-ranktest). The occurrenceof overt leukemia in SCID mice
was was highly
a
significant predictor of patient relapse. The
estimated instantaneous risk of relapse for patients whose
leukemic cells caused
overt leukemia in SCID mice was 21.5fold greater than that for the remaining patients. Thus,
growth of human leukemic cells in SCID mica is a strong
and independentpredictor of relapsein patients with newly
diagnosed high-risk B-lineageacute lymphoblastic leukemia.
Q 1995 by The American Society of Hematology.
A
Pathologic Specimens) in accordance with DHHS guidelines. Patient
characteristics are detailed in Table 1. All 42 patients had high-risk
ALL, as identified by age (ie, < l or = l 0 years), white blood cell
count (WBC; ie, z50,000/pL), or cytogenetics (ie, presence of a
4; 1 1 , 1; 11, or 9;22 translocation) according to the uniform highrisk criteria of the National Cancer Institute (NCI)/Cancer Treatment
Evaluation Program (CTEP) Workshop for B-lineage ALL patients.
All patients received intensive treatment for high-risk ALL. CCG
patients were treated with a Berlin-Frankfurt-Munster (BFM)-based
or New York-based intensive multiagent chemotherapy regimen,”.I6
whereas POG patients were treated with primarily antimetabolitebased intensive chemotherapy.*’ Fourteen patients were treated on
CCG regimens and 28 patients on FOG regimens. Follow-up on
patients still surviving without the occurrence of leukemic relapse
events ranges from 6 to 66 months, with a median follow-up of 32
months.
Laboratory studies. Specimens for this study were obtained by
routine diagnostic bonemarrow aspirates. Highly blast enriched
CUTE LYMPHOBLASTIC leukemia (ALL) is the
most common childhood malignancy, comprising
about 25% of all pediatric cancer
Eighty-five percent of cases arise in B-progenitor cells and can be identified
as B-lineage ALL by diagnostic immunophenotyping.Is2Approximately 70% of these patients are cured with intensive
multiagent chemotherapy. Hence, current research is aimed
at improving therapy for children who are at risk for relapse,
while minimizing treatment intensity in those who have a
favorable prognosis. The risk-directed approach has the potential to decrease late effects of treatment (a major concern
in these young patients) while intensifying treatment in appropriate cases to improve cure rates even further. Clearly,
accurate methods of identifying prognostic features at diagnosis are crucial to avoid unnecessarily intensive treatment
or inadequate therapy.
We and others have used mutant C.B.17 mice with severe
combined immunodeficiency (SCID) as a model system to
examine the in vivo homing, engraftment, and growth patterns of n o d and malignant human hematopoietic cells.%”
These mice lack functional T and B cells and are unable to
reject allogeneic or xenogeneic grafts.23We compared the
ability of leukemic cells from 42 children with newly diagnosed high-risk B-lineage ALL to cause leukemia in SCID
mice and assessed these findings in relation to the patients’s
response to treatment. Our findings suggest that the outgrowth of a patient’s leukemic cells in the SCID mouse is
a strong and independent predictor of relapse after chemotherapy.
MATERIALS AND METHODS
Patient population. Forty-two children with newly diagnosed
untreated B-lineage ALL treated at member institutions of the Childrens Cancer Group (CCG) or Pediatric Oncology Group (POG)
were studied. Informed consent for treatment was obtained from
parents, patients, or both based on Department of Health and Human
Services (DHHS) guidelines. Bone marrow specimens were used in
the SCID mouse assay system under the exemption category (45
CFR Part 46.101;b category #4 Existing Data; Records review;
Blood, Vol 85, No 4 (February 15). 1995: pp 873-878
From rhe University of Minnesota Biotherapy Program, the Departments of Therapeutic Radiology and Pediatrics, Minneapolis,
MN; the Childrens Cancer Group, Arcadia, CA; and the Pediatric
Oncology Group, Chicago.
Submitted September 8, 1994; accepted November 2, 1994.
Supported in partby research grants (CA-60437,CA-51425,
CA-21737, CA-4211 I , CA-42633,CA-21765[COREl,CA-30969,
CA-31566, and CA-13539)from the National Cancer Institute, Department of Health and Human Services, the National Cancer Foundation, and the American Lebanese Syrian Associated Charities
(ALSAC). F.M.U. is a Stohlman Scholar of the LPukemia Society of
America.
This is publication #l30 from the Tumor ImmunologyLaboratory,
University of Minnesota.
Address reprint requests to Fatih M. Uckun, MD, PhD, CCG
ALL Biology Reference Laboratory and University of Minnesota
Biotherapy Program, 2685 Patton Rd, Roseville, MN 551 13.
The publication costs of this article were defrayed in p a n by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
Q 1995 by The American Society of Hematology.
ooW-4971/95/8504-0031$3.00/0
873
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874
UCKUN ET AL
Table 1. Characteristics of 42 High-Risk B-LineageALL Patients
Whose Leukemic Cells Were Studied in the SClD Mouse System
Characteristic
Distribution
No. (%)
Age (yr)
<l
1-9
210
Male sex
WBC 2 5 0 X 1 0 ~ ~
DNA index (n = 28)
21.16
<1.16
Cytogenetics (n = 28)
Normal
t(9;22), or t(411) or t(1;19)
Splenomegaly
Hepatomegaly
CNS leukemia
Outcome in SClD mice
Leukemia detectable by histopathology
Occult leukemia
No evidence of leukemia
6 (21)
22 (79)
17 (40)
14 (33)
3 (7)
23 (55)
11 (26)
8 (19)
Occult leukemia is ~ 1 0 %
of cells from SClD mouse bone marrow,
spleen, or liver positive for human CD19 antigen by flow cytometry
or human &globin gene sequences detected by PCR analysis of DNA
from SClD mouse bone marrow, liver, spleen, or brain.
mononuclear cell fractions containing more than 90% leukemic cells
were isolated by Ficoll Hypaque density gradient separation. Immunophenotyping was performed on these fractions by indirect immunofluorescence andflow cytometry using appropriate monoclonal
antibodies directed against CD5, CD7, CDlO, CD13, CD19, CD20,
CD21, CD22, CD24, CD34, sIg, and MHC-11, as previously described.” Cytogenetic studies and DNA flow cytometry were performed, as previously reported.’
All SCID mice (age, 7 to 10 weeks; sex, female) were produced
by specific pathogen-free (SPF) CB-l7 scidscid breeders and maintained in an SPF environment in Micro-Isolator cages (Lab Products,
Inc. Maywood, NY), as previously reported.2426Mice (1 to 3, depending on the availability of sufficient cell numbers) were inoculated with 5 X lo6 leukemic cells via tail vein injections. Mice were
killed at 12 weeks or when they became moribund as a result of
disseminated leukemia. Mice were necropsied at the time of death or
euthanization, and histopathology, flow cytometry, and polymerase
chain reaction (PCR) analyses were performed to assess their burden
of human leukemia cells, as previously reported.2426For each mouse,
multiple tissues, including brain, lung, heart, thymus, liver, kidney,
spleen, gut, pancreas, urinary bladder, uterus, ovary, sagittal sections
of a femur, and several vertebrae, were histologically evaluated. All
histopathologic studies of SCID mouse tissues were performed by
a veterinary pathologist (ie, R.G.) without any knowledge of patient
outcome. Multiparameter flow cytometric analyses of single-cell suspensions from SCID mouse bone marrow, liver, and spleen were
performed using two-color immunofluorescence staining techniques
and pairwise combinations of a selected panel of fluorochromelabeled (fluorescein isothiocyanate W C ] or phycoerythrin [PE])
monoclonal antibodies to identify human B-lineage ALL blasts.w26
The following monoclonal antibodies were included in the analyses:
B43 (anti-CD19)-PE, Leu12 (anti-CD19)-FITC, J5 (anti-CDlO)-PE,
Leu16 (anti-CD20)-FITC, 9.4 (anti-CD45)-PE, 9.4 (anti-CD45)FITC, and 2C3 (anti-1gM)-FITC. Human DNA was detected by
amplifying a 110-bp fragment from the first exon of the human 0-
globin gene using two 20-base oligonucleotide primers, PC03 and
that flank the region to be amplified, as previously described
in
All
evaluations of SCID mouse tissues were conducted
in a prospective fashion andthey were blinded to knowledge of
treatment outcome.
Statistical analysis. Possible associations of leukemic cell outgrowth in SCID mice with clinical, demographic, and laboratory
features of thepatients from whom cells were derived were examined
by using x’ analyses of categorical groupings. The primary outcome
measure was event-free survival (EFS), definedas the time from
study entry to the first major event (failure to achieve remission,
relapse at any site, or death). For patients who did not experience
an event, EFS was defined as the time to last follow-up and treated
as a censored life table observation. Estimates of EFS were based
on the Kaplan-Meier life table method for censored data.35Standard
errors for the life table estimates were calculated using Greenwood’s
formula. Comparisons of outcome between patient subsets were performed by the log rank test for univariate comparisons and for those
that were stratified on a single additional fa~tor.~’,~’
Calculations of
the relative event rate (ie, hazard rate) used the O/E method for
univariate or stratified analysis and the exponent of the regression
coefficient procedure for the proportional hazards regression.38Confidence intervals (95%) were calculated using the log-log transformationof the survival function together with Greenwood’s formula
because EFS outcome for one patient group was almost 100%.Multivariate analysis was performed using the Cox proportional hazards
regression model for all factors with reasonably complete data (ie,
leukemic cell growth in SCID mice, WBC, age, sex, hepatomegaly,
splenomegaly, platelet count, hemoglobin level, and central nervous
system [CNS] disease at diagnosis). All statistical analyses were
performed by the Group Statistician of the Childrens Cancer Group
(ie, H.S.).
PCO4.
RESULTS
Human leukemiacells are found in aggregates in the SCID
mouse organs other than bone marrow and they almost always destroy the normal organ architecture. Therefore, extramedullary human leukemia is easily detected in SCID mouse
tissues. The sensitivity of histopathologic determinations in
SCID mouse bone marrow is comparable with that of standard morphologic determinations ofleukemic cell burden in
human bone marrows. O n e percent to 5% involvement is
readily detectable; sometimes even a 0.1 % involvement can
be detected by histopathology if leukemic cells are found in
aggregates. In the present study, a marked interpatient variation existed in the extent of leukemic cell engraftment and
outgrowth in SCID mice challenged with primary bonemarrow blast cells from children with high-risk B-lineage ALL.
However, there were no intrapatient inconsistencies; in each
case in which cells were inoculated into two or more mice,
each of the animals exhibited the same pattern of leukemic
cell growth (or lack thereof). No histopathologic evidence
of leukemia was found in SCID mice inoculated with leukemic cells from 19 of 42 (45%) patients (Table 1). However,
in 11 of these 19 cases, occult human leukemia could be
detected in SCID mouse tissues by multiparameter flow cytometric analysis of bone marrow, liver, and spleen mononuclear cell suspensions for CD19+CDlO+”sIgM-B-lineage
ALL cells (2 cases only; the fraction of flow cytometrically
detectable human B-lineage ALL cells among the mononuclear cells obtained from bone marrow, liver, or spleen of
these mice did not exceed 10%)
and/or PCR analysis of
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HUMAN ALL IN SCID
875
Table 2. Relationship Between Patients' Presenting Clinicalor Laboratory Features and Development
of Histopathoiogically Detectable Leukemia in SCiD
Mice
~~
~
~~~
Age (yr)
Splenomegaly
Hepatomegaly
CNS involvement
WBC ( ~ 1 0 9 1 ~ )
(69.6%) 16
Platelet count (xlOg/L)*
Hgb level (g/dL)*
Cytogeneticst
(N = 191
~
~~
1 (5.3%)
2 (8.7%)
(65.2%)
15
.21 8 (42.1%)
(52.6%)
6 10
(26.1%)
Male .32 (63.2%)
12 1 1 (47.8%)
Female
(52.2%)
12
7 (36.8%)
(56.5%)
13
.02 4 (21.1%)
Absent
(78.9%)
15
(43.5%)
10
Present
8 (34.8%)
.83 6 (31.6%)
Absent
(68.4%)
13
(65.2%)
15
Present
2 (8.7%)
.67
1 (5.3%)
18 (94.7%)
Absent
(91.3%)
21
<50
7 (30.4%)
.66
7 (36.8%)
2 50
12 (63.2%)
9 (50.0%)
<50
(73.9%)
17
9 (50.0%)
250
6 (26.1%)
.04 6 (33.3%)
<8
(65.2%)
15
2 8 (66.7%)
12
8 (34.8%)
1 1 (57.9%)
<1.16
21.16
1 (5.3%)
0 (0.0%)
7 (36.8%)
Unknown
7 (30.4%)
Normal
3 (13.0%) .83
3 (15.8%)
8 142.1%)
14 (60.8%)
Pseudodiploid
Unknown
8 (42.1%)
6 (26.1%)
~
~
<l
1-9
=l0
Sex
DNA indext
(69.6%)
~~
P
Value
SCID- Patients
SCID' Patients
(N = 231
Category
Features
-~
16
.l 1
.88
Diagnostic values for Hgb level or platelet count were not available for one of the SCID- patients.
t DNA flow cytometry and karyotyping were each performed in 28 cases.
DNA from bone marrow, liver, spleen, or brain for human
@-globingene sequences (all 11 cases), which has previously
been shown to detect a 0.1% human leukemic cell contamination in SCID mouse tissuesz5 (Table 1). By comparison,
leukemic cells from the remaining 23 (55%) patients caused
histopathologically detectable overt leukemia in SCID mice
(Table 1). In these mice, bone marrow was always involved,
whereas the involvement of other organs showed interpatient
variations. Bone marrow, spleen, and ovarian involvement
was manifested as replacement of normal tissue elements by
diffuse sheets of densely packed leukemic cells with up to
20 mitotic figures per high power field. When involved,
livers showed infiltration in the subcapsular region, portal
spaces, and sinusoids. Infiltrated kidneys had variably sized
cortical, interstitial, and perivascular accumulations of leukemic cells and often had large masses in the pelvic fat. The
leptomeninges of the brain contained thin rafts of leukemic
cells. Affected lungs contained a light infiltrate in the alveolar septa, small rafts of leukemic cells in the intrapulmonary
veins, or perivascular cuffs of leukemic cells. Multiparameter flow cytometric analyses of bone marrow mononuclear
cells from SCID mice with histopathologic evidence of marTOW involvement confirmed the abundance of a 1 0 + "
CD19+CD45+sIgM- human B-lineage ALL cells. PCR amplification of a 110-bp DNA fragment from the first exon of
the human @-globingene confirmed the presence of human
origin DNA in bone marrow, liver, spleen, and brain of SCID
mice with disseminated B-lineage ALL.
When we examined the possible association of leukemic
cell growth in SCID mice with high-risk ALL patients' clini-
cal, demographic, and laboratory features, we found that
leukemic cells from patients with splenomegaly (P = .02)
and with low hemoglobin levels (P = .W)were slightly
more likely to cause histopathologically detectable leukemia
in SCID mice (Table 2). By comparison, age, sex, hepatomegaly, CNS disease, leukocyte count, platelet count, DNA
index, or specific chromosomal translocations did not correlate significantly with leukemic cell growth in SCID mice
(Table 2).
The 23 patients whose leukemic cells caused histopathologically detectable leukemia in SCID mice had a significantly higher failure rate than those patients whose leukemic
cells did not (N = 19; 14 relapses and one induction failure
v 1 relapse; log rank test, P = .OOOl; univariate life table
estimated relative hazard, 21.5). Consequently, the former
group had a significantly worse 5-year EFS (29.5% v 94.7%;
95% confidence intervals, 11.2% to 50.7% v 68.1% to
99.2%; P = .OOO1 by log rank test; Fig 1). Stratified log
rank analyses were conducted to adjust individually for potentially important clinical and laboratory features, such as
W C , cytogenetics, age, sex, hepatomegaly, splenomegaly,
CNS involvement, platelet count, hemoglobin, immunophenotype (ie, percent positivity of leukemic cells for surface
CD10, CD19, CD24, or CD34 antigen expression), and DNA
index. Histopathologically detectable leukemia in SCID
mice remained a verystroKg predictor of patient relapse after
adjusting individually for the effects of each of these factors.
All significance levels for the SCID model predictor were
less than or equal to .006.Multivariate Cox regression analysis was also performed and only two factors, namely histo-
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UCKUN ET AL
876
1.oo
only byflow cytometry and/or PCR or failed to engraft in
SCID mouse tissues. Notably, the ability of leukemic cells
Q,
to cause histopathologically detectable leukemia in SCID
9?
mice was associated with poor EFS in patients from whom
5
the cells were obtained. Adjustment for numerous traditional
E 0.75
prognostic factors in bivariate stratified log-rank analyses
W
and multivariate analysis using the Cox proportional hazards
regression
modelhadvery
little attenuating effect on the
.prognostic significance of occurrence of overt leukemia in
.->
2 0.50
LeLkemhin
SCID mice for this patient population with high-risk ALL.
SClD mice,
Biologic data generated in the SCID mouse model system
may thus improve our ability to predict treatment response
C
0
.-c
in high-risk B-lineage ALL, permitting accurate segregation
of patients into distinct prognostic subgroups.
0.25
Histopathologic examinations showed no evidence of leuP=0.0001
kemic
infiltrates in the organs of SCID mice challenged with
a
primary blasts from19of
the 42 patients. In 11 of these
I
I
I
I
I
I
I
I
I
I
patients, occult leukemia cells were detected by multipa0.0
1
2
3
4
5
rameter flow cytometry and/or PCR in SCID mouse bone
marrow, liver, spleen, andor brain. However, none of these
Years F m Study Entry
11 patients have relapsed. Thus, occult leukemia in SCID
mice, in the absence of histopathologic evidence of leukemic
Fig 1. EFSofB-lineage ALL patients in relation to development
of human Iaukemiain SClD mice inoculatedwith their leukemic cells.
infiltrates in any of the SCID mouse organs, did not have
Patients whose leukemic cells didnot cause histopathologically dean apparent prognostic impact. Larger studies with longer
tectable leukemia in
SClD mice In = 19) fared significantly better than
follow-up are needed to determine whether this subgroup of
those whose leukemic cells produced histopathologically detectable
patients may be at increased risk of late relapses.
leukemia in this model (n = 23). Tick marks on the curves indicate
the last follow-up time for patients who have not experienceda disThe reasons for the observed interpatient heterogeneity
ease event. The one relapse in
the group of patients whose leukemic
and prognostic impact of leukemic cell growth in SCID mice
cells failed to causeleukemiain SClD mice occurred at 6 months
remain unclear. Kamel-Reid et a1," Cesano etal,"and
postdiagnosis (95% confidence interval estimate for EFS
= 68.1%.
Uckun et a124,2s
have reported that leukemic B-cell precursors
99.2%). The latest relapse to date for the group of patients whose
from established pre-B ALL cell lines cause disseminated
leukemic cells caused leukemiain SClD mice was at 30 months. For
this group of patients, the 95% confidence intervals for EFSat 6,18,
leukemia in SCID mice. Recently, Kamel-Reid et a13' also
and 30 months of follow-up are 46.6%to 84.2%. 26.8% to 66.1%. and
reported that leukemic cells from five patients with relapsed
11.2% to 50.7%. respectively.Theestimatedmedian
EFS for the
B-lineage ALL proliferated rapidlyin SCID mouse bone
group whose leukemic calls caused overt leukemia in SClD mice is
marrow, spleen, thymus, and peripheral organs, leading to
9.5 months.
morbidity and mortality. It is possible that the SCID mouse
model system favors the in vivo expansion of drug resistant
blast populations and/or highly clonogenic blast populations
pathologically detectable leukemic cell growth in SCID mice
capable of rapid self-renewal and clonal expansion after es( P = .004) and hepatomegaly ( P = .05) had predictive value
caping the lethal actions of multiagent chemotherapy.
in this subset of poor prognosis patients.
Of the numerous clinical and biologic factors reported to
Unlike the development of histopathologically detectable
be of prognostic value in assessing the risk of relapse after
leukemia in SCID mice, detection of occult leukemia in the
contemporary therapy for ALL,'.',9.1',,'7.'8
the most consistent
absence of histopathologic evidence of leukemic infiltrates
are age and leukocyte count at diagnosis. However, these
in any of the SCID mouse organs did not have an apparent
features alone or in combination are not sufficient to identify
prognostic impact. None of the 11 patients whose leukemic
all patients at increased risk of relapse.' Genetic features
cells caused occult leukemia in SCID mice as detected by
of leukemic cells (tumor cell DNA content or ploidy and
multiparameter flow cytometry and/or PCR have relapsed
karyotype) have also been shown to provide additional progand all remain alive and disease free at 23 to 60 months
nostic inf~rmation.~
Flow cytometric assessment of ploidy is
(median, 42 months) after diagnosis.
rapid, relatively easy and inexpensive, and produces accurate
results in almost allcases.' Leukemic cell hyperdiploidy
DISCUSSION
(DNA index 2 I . 16) identifies a subgroup of B-lineage ALL
patients withan estimated 4-year disease-free survival of
We found a pronounced variation in the ability of leukegreater than 90%in the context of antimetabolite-based treatmic cells from children with newly diagnosed high-risk Bm
e ~ ~However,
t.~
ploidy is useful in identifying only the 20%
lineage ALL to engraft and cause leukemia in SCID mice.
of patients who are at lowrisk of treatment failure and cannot
Leukemic cells from some patients caused disseminated hupredict a high risk of relapse.' The SCID mouse system
man leukemia in SCID mice, as detected by histopathology
described here eliminates some of the problems associated
and confirmed by flow cytometry as well as PCR. Leukemic
with other prognostic tools, appears to complement the progcells from other patients produced occult leukemia detectable
2
P
8
g
e
"i
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
HUMAN ALL IN SCID MICE
nostic value of conventional risk features such as age and
leukocyte count, and holds potential for large-scale evaluation of newly diagnosed cases of B-lineage ALL. Results
from this assay could be very useful for determining the
intensity of further therapy after remission induction in highrisk B-lineage ALL. In addition to providing information
of independent prognostic value for patients with high-risk
features, this SCID mouse model of human B-lineage ALL
may also permit assessment of the “in vivo” activity of
individual therapeutic agents against the patient’s leukemic
cells. Such information could be highly useful for planning
the components of intensification as well as maintenance
chemotherapy in high-risk B-lineage ALL.
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1995 85: 873-878
Leukemic cell growth in SCID mice as a predictor of relapse in highrisk B-lineage acute lymphoblastic leukemia
FM Uckun, H Sather, G Reaman, J Shuster, V Land, M Trigg, R Gunther, L Chelstrom, A Bleyer
and P Gaynon
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