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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2364-2371
NEOPLASIA
Cell cycle regulatory gene abnormalities are important
determinants of leukemogenesis and disease biology in adult acute
lymphoblastic leukemia
Wendy Stock,
Tsuong Tsai,
Carla Golden,
Cathy Rankin,
Dorrie Sher,
Marilyn L. Slovak,
Maria G. Pallavicini,
Jerald P. Radich, and
David H. Boldt
From the Department of Medicine, University of Illinois at Chicago,
Chicago, IL; the Department of Medicine, University of Texas Health
Science Center, the Audie L. Murphy Memorial Veterans Hospital, and the
Southwest Oncology Group Leukemia Biology and Cytogenetics Programs,
San Antonio, TX; the Cancer Center, University of California, San
Francisco, CA; the Department of Hematology/Oncology, Children's
Hospital, Oakland, CA; the Southwest Oncology Group Statistical Center
and the Program in Genetics, Clinical Research Division, Fred
Hutchinson Cancer Research Center, Seattle, WA; and the Department of
Cytogenetics, City of Hope National Medical Center, Duarte, CA.
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Abstract |
To test the hypothesis that cell cycle regulatory gene abnormalities
are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a
Southwest Oncology Group protocol for abnormalities of the genes,
retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant
expression occurred in 33 (85%) patients in the following frequencies:
Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had
abnormalities in 2 or more genes. Outcomes were compared in patients
with 0 to 1 abnormality versus patients with multiple abnormalities.
The 2 groups did not differ in a large number of clinical and
laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%,
respectively). Patients with 0 to 1 abnormality had a median survival
time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months
(n = 13; 95% CI, 4-12 months) for those with multiple
abnormalities (P < .01). Stem cells (CD34+lin )
were isolated from adult ALL bone marrows and tested for p16(INK4A)
expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and
sorted stem cells lacked p16(INK4A) expression. In 2 other patients
only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16
expression was present in the CD34+ lin compartment in 95%
(median) of 9 patients whose lymphoblasts expressed p16(INK4A).
Therefore, cell cycle regulatory gene abnormalities are frequently
present in adult ALL lymphoblasts, and they may be important
determinants of disease outcome. The presence of these abnormalities in
the stem compartment suggests that they contribute to leukemogenesis.
Eradication of the stem cell subset harboring these abnormalities may
be important to achieve cure.
(Blood. 2000;95:2364-2371)
© 2000 by The American Society of Hematology.
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Introduction |
Substantial incremental success in the treatment and
management of childhood acute lymphoblastic leukemia (ALL) over the
past 3 decades has not impacted significantly on the prognoses of
adults with ALL. Whereas 70% to 75% of children with ALL now can
expect to be cured of their disease, the same expectation pertains to no more than 30% to 35% of adults.1,2 Compared to
children, adults with ALL are more refractory to initial chemotherapy
and more likely to develop resistance to ongoing treatment. These observations suggest that different etiologic events contribute to the
pathogenesis of childhood and adult ALL. Although differences have been
described in morphologic and immunologic characteristics and, more
important, in the distribution of molecular genetic abnormalities, no
single feature among these variables appears to account for the marked
differences in clinical and biologic behaviors. For adult patients with
ALL, age, white blood cells (WBC), and cytogenetics are
well-established prognostic factors. However, many adult patients do
not have any of these distinguishing features. Therefore, there is a
need to identify other factors that may aid in assessing prognosis in
this difficult group of patients. Recently many investigators have
reported abnormalities of tumor suppressor or cell cycle regulatory
genes in patients with ALL.3,4 Among the genes in this
category most frequently altered in adult ALL are p16(INK4A),
p15(INK4B), the retinoblastoma tumor suppressor gene (Rb), and p53.
Increasing evidence indicates that abnormalities of these genes are
common, perhaps universal, in adult ALL and that substantial numbers of
patients have abnormalities of more than 1 gene in this category.
Although single tumor suppressor gene abnormalities have not generally
correlated with prognosis, we hypothesized that multiple abnormalities
of cell cycle regulatory genes may strongly influence clinical outcome
in adult ALL. Furthermore, we suggest that the acquisition of these
abnormalities by clonal neoplastic bone marrow precursor cells in the
stem cell compartment is an important prerequisite of the leukemogenic
process. These hypotheses are based on observations that progression is
often associated with acquisition by the neoplastic clone of multiple
genetic aberrations prevalent in leukemias with poor prognoses. To test
these concepts we analyzed expression of a group of cell cycle
regulatory genes, Rb, p53, p16(INK4A), and p15(INK4B), in a cohort of
adult patients with ALL uniformly treated on a Southwest Oncology Group
(SWOG) protocol with mature follow-up. In addition, to determine
whether p16(INK4A) abnormalities were present in stem cells, we
assessed alterations of p16(INK4A) in adult ALL bone marrow sorted into
stem, early, and late lineage committed precursor compartments.
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Materials and methods |
Patient specimens and clinical protocols
Samples were analyzed from 42 patients with de novo adult ALL
registered on treatment study S8417/19: Evaluation of 2 Consolidation Regimens in the Treatment of Adult Acute Lymphoblastic
Leukemia.5 This protocol was open to patient accrual from
June 14, 1985 to November 15, 1991. Eligibility criteria included age
15 years or older, absolute infiltration of the bone marrow (determined by % blasts × cellularity) with 50% lymphoblasts or 30% to
40% lymphoblasts with evidence of progressive disease, and no prior leukemia treatment. The diagnosis of ALL was confirmed by central review of morphology and cytochemistry according to the previously described SWOG modification of the French-American-British (FAB) criteria.6 The treatment included 5-drug induction with an L10M regimen,7 followed by randomization to consolidation
therapy with either the L10M regimen or alternative chemotherapy with daunorubicin, cytosine arabinoside, 6-thioguanine, L-asparaginase, and
methotrexate. After consolidation, all patients received uniform, multi-drug L10M maintenance therapy. No difference in outcomes was
observed for patients registered to the 2 different consolidation arms.5 Central immunophenotype determinations were
performed on pretreatment specimens at the SWOG Lymphoid Leukemia
Reference Laboratory at the University of Texas Health Science Center
at San Antonio by methods previously reported.8 Specimens
were sent to the central laboratory by overnight courier. Samples in transit for more than 24 hours or samples with viability less than 90%
were considered inevaluable. Cytogenetic evaluations were not required
for patients registered to protocol S8417. Cytogenetic results
performed by patients' local institutions were reviewed when possible
by the SWOG Cytogenetics Committee. As a result, data were available
for 16 subjects in this analysis. Furthermore, the presence or absence
of the p190 or p210 BCR-ABL fusion genes was assessed by reverse
transcription-polymerase chain reaction (RT-PCR) in 34 of these
subjects.9
The stem cell sorting experiments were performed using 16 specimens
from patients registered on protocol S9400: Treatment of Adult Acute
Lymphoblastic Leukemia: A Phase II Trial of an Induction Regimen
Including PEG-L-Asparaginase, in Previously Untreated Patients,
Followed by Allogeneic Bone Marrow Transplantation or Further
Chemotherapy in First Complete Remission. This ongoing protocol was
open to patient accrual on July 15, 1995. Eligibility, diagnostic
criteria, and immunophenotyping were performed as described above.
Immunodetection of Rb and p53 proteins
Assessments of lymphoblast expression of pRb and p53 were performed
as previously described.10 Immunoblotting was used to analyze lymphoblast lysates for Rb or p53 protein expression. Limiting
dilution experiments indicated that the technique was sufficiently
sensitive to permit detection of pRB or p53 in as little as 1 µg
lysate protein from cryopreserved normal activated peripheral blood
lymphocytes (data not shown). All patient samples that lacked pRB or
p53 expression by this technique were subsequently subjected to
additional analyses by electrophoresis and silver staining to ensure
integrity of the protein composition and to exclude the possibility
that absence of pRb or p53 expression represented sample degradation.
In addition, a nonisotopic RNase cleavage assay based on techniques
originally described by Myers et al11 and Winter et
al12 was used to analyze p53 overexpressing specimens for
point mutations in exons 5 to 9 of the p53 gene. (Mismatch Detect Point
Mutation Screening Kit; Ambion, Austin, TX). Point mutations were
confirmed in 61% of patients with aberrant p53 expression identified
by immunoblotting.10
Analyses for p16(INK4A) abnormalities
Two methods were used to analyze samples for p16(INK4A)
abnormalities. The first was Southern blot analysis of p15, p16
abnormalities. DNA was extracted using standard methods.13
Approximately 10 µg extracted DNA from blood and bone marrow samples
was enzymatically digested with HindIII (New England Biolabs;
Beverly, MA). Digests were electrophoresed on standard agarose gels and
transferred to a nylon membrane (Gene-Screen Plus; Dupont/NEN,
Wilmington, DE). Southern blot analysis was performed by hybridization
of digests to a 1 kb Xbal/Xhol cDNA probe that
recognizes exon 2 of p15 and exons 1 to 3 of p16. All blots were
hybridized sequentially to a genomic probe (2.2 × X) from the
TAL1 gene, which served as a control for DNA
integrity.14 Blots were washed to a final stringency of
0.1 × saturated sodium citrate (SSC) and 1% sodium dodecyl
sulfate at 65°C before autoradiography. Blots were exposed to film
for 3 to 8 days.
The second method used to analyze samples for p16(INK4A) abnormalities
was PCR. In some cases, PCR analysis was carried out to determine
specific deletions of the various exons of the p16 gene as described
previously for exons 1 and 215 and for exon 3.16 The PCR-amplified products were fractionated on a 2%
agarose gel and visualized by ethidium bromide staining.
Flow cytometric analyses and sorting of ALL marrows and blood
specimens
Cryopreserved specimens were sent to the Cancer Center at the
University of California, San Francisco, for immunostaining and cell
sorting. Specimens were sorted into 3 differentiation compartments as
previously described17,18 (Figure 1).
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| Fig 1.
Multivariate flow cytometric analysis of specimen 15.
(A) Bivariate light scatter distribution. R1 is the gate used to
discriminate nucleated cells. (B) PI fluorescence intensity of R1 gated
cells. R2 defines viable cells contained within the R1 gate. (C)
Isotype FITC- and PE-labeled control and (D) CD34-linked
immunofluorescence (abscissa) versus lineage markers (ordinate). Sort
gates: R5 (CD34+lin), R6 (CD34+lin+), R7 (CD34-lin+).
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Flow cytometric analysis and sorting were performed on a FACStar Plus
Cell Sorter (Becton Dickinson, Mountain View, CA) equipped with 2 argon-ion lasers (Coherent, Palo Alto, CA) tuned to 488' nm
(primary laser) and to 351-364 nm (secondary laser). Small debris and
cell aggregates were excluded by forward and side light scatter
measurements, and doublets were excluded using forward scatter pulse
width and area measurements. Fluorescein isothiocyanate (FITC) and
phycoerythrin (PE)-linked immunofluorescence emissions from the primary
laser excitation were collected through 530 nm (± 30 nm) and 575 nm (± 26 nm) bandpass filters, respectively. Propidium iodide (PI)
emissions from secondary laser excitation were collected through a
620-nm long-pass filter.
The frequency of discriminated subpopulations was estimated as
described previously.17 The number of PI-negative cells
within the light scatter gate (see Figures 1A and 1B) in each specimen constituted the denominator value used for subpopulation estimation. The average frequency of viable cells in the 24 specimens approximated 85% (range 50% to 99%). Quadrant markers were set at the first decade of the FITC and PE fluorescence intensities, and photo multiplier tube voltages were adjusted so that gated specific regions
contained less than 0.01% of viable nucleated cells from the isotype
control sample. Fluorescence compensation was performed using single
fluorochrome-positive controls. Rectangular regions within the positive
quadrant boundaries were used to sort 3 compartments: CD34 33+38+19+
(CD34lin+),
CD34+33+38+19+
(CD34+lin+) and
CD34+333819
(CD34+lin). The stringent sort regions, set 5% to 15% away
from the quadrant boundaries, were used to minimize contamination from adjacent cell subpopulations during the sort. Cells from these regions
were sorted into wells on Teflon-coated glass microscope slides (DuPont, Wilmington, DE) using a 2-drop packet in Normal-C mode
to ensure maximal purity (ie, more than 98%), and cells fixed with
Carnoy's (methanol/glacial acetic acid; 3/1, vol/vol). The sorted
cells were dried on a slide warmer at 37°C for 30 minutes then
fixed again with fresh Carnoy's.
Immunocytochemical determination of p16(INK4A)
Expression of p16(INK4A) in the sorted bone marrow compartments was
assessed by immunocytochemistry. Briefly, slides containing sorted
cells were incubated overnight with p16(INK4A) monoclonal mouse
antibody 1:100 (Neomarkers p16-Ab-1; Neomarkers, Fremont, CA) and then
incubated with biotinylated antimouse antibody 1:100 (Vector
Laboratories, Burlingame, CA). Bound antibody was detected with the
Vectastain ABC kit (Vector Laboratories). Slides were washed, incubated
with osmium, fixed, and mounted for viewing by light microscopy.
Statistical analysis
Collection and quality control of patient pretreatment and outcome
data were performed according to standard SWOG procedures. P
values for comparisons of continuous variables between groups of
patients were 2-tailed and based on the Wilcoxon rank sum
test.19 P values for dichotomous variables were
based on the Fisher exact test20,21 using direct
calculation to avoid large sample approximations. The remaining
P values were based on the Pearson chi-squared
test22 using direct calculation of P values to
avoid large sample approximations. Overall survival was measured from
the day of registration in the study until death from any cause and was
censored only for patients known to be alive at last contact.
Disease-free survival (DFS) was measured from the day that complete
response (CR) was established until either relapse of ALL or death
without relapse, and it was censored only for patients who were alive
without evidence of relapse at the last follow-up. Distributions of
overall survival and DFS curves were estimated by the method of Kaplan
and Meier.23 Comparisons of overall survival or DFS between
groups were based on the log-rank test.24 Comparisons
adjusted for significant prognostic factors were based on Cox
regression models23 for CR rates and on proportional
hazards regression models24 for survival and DFS.
| |
Results |
Patient specimens
Cryopreserved bone marrow or peripheral blood specimens (greater
than or equal to 80% lymphoblasts) were used to analyze 42 de novo
adult patients with ALL from protocol S8417 for abnormalities of the
cell cycle regulatory genes, Rb, p53, p15(INK4B), and p16(INK4A). These
42 patients were all those remaining in the S8417 repository with
sufficient biologic material for the cell cycle regulatory gene
analyses. Sufficient cells for assessment of all genes were available
from 39 patients and for 2 genes from 3 patients. Characteristics of
these patients and leukemic specimens are given in Table
1. The patients in the current study are
similar to the entire population registered to S8417 for FAB
classification, blast cell lineage, co-expression of myeloid antigens,
and blast cell expression of the CD34 stem cell antigen. In the current
study, 27 of 42 (64%) patients achieved CR, which is similar to the CR
frequency (62%) of all patients enrolled in the S8417
study.5 However, in previous analyses of S8417, age and WBC
or peripheral lymphocyte count were prognostic for overall and
relapse-free survival.5 The values of these parameters
indicate that these 42 patients in the current study represent a poor
prognostic subset.
Incidence of cell cycle regulatory gene abnormalities
The incidence of cell cycle regulatory gene abnormalities in the
study cohort was high (Table 1). Aberrant expression of Rb, p16(INK4A),
or p53 was identified in 21 of 41 (51%), 17 of 42 (41%), and 10 of 39 (26%), respectively. Twenty-nine specimens analyzed by Southern
blotting were evaluable for p15(INK4B) deletions or rearrangements.
Deletions were identified in 9 of 29 (31%) patients. Because in each
of these 9 patients p15 was co-deleted with p16(INK4A), aberrant
p15(INK4B) expression has not been analyzed separately. The incidences
of Rb and p53 abnormalities are similar to those we reported previously
in a larger subset of S8417 patients.9 The frequency of
specimens with 1 or more abnormal cell cycle regulatory gene in the 39 patients who were evaluated for the 3 genes is shown in Table
2. At least 1 abnormality of either Rb,
p53, or p16(INK4A) occurred in 33 of 39 (85%) patients. Of these 33 patients, 20 (61%) had abnormalities of only 1 cell cycle gene. Twelve
patients had abnormalities of 2 cell cycle genes, and 1 had
abnormalities of Rb, p53, and p16. Thus 33% of all patients had
abnormalities of multiple cell cycle regulatory genes.
We used Southern blotting, PCR, or both to test for p16(INK4A) gene
deletions or rearrangements. In the 39 patients analyzed for this
study, both Southern blotting and PCR were preformed in 14, Southern
blotting alone in 15, and PCR alone in 10. Sample availability
precluded performing both assays in every patient. Overall,
abnormalities of p16(INK4A) expression were detected in 17 patients
(Table 3). Gene deletions/rearrangements
were identified in 13 of 29 (45%) patients by Southern blotting and in
13 of 24 (54%) patients by PCR (data not shown).
In patients analyzed by both Southern blotting and PCR, there was good
concordance between results. Concordant results were observed in 12 of
14 patients. In 3 patients Southern analyses indicated an intact
p16(INK4A) gene and PCR detected the appropriate exons 1 and 2 (not
shown). Table 3 gives results of Southern blotting and PCR analyses in
the 17 patients with aberrant p16(INK4A) expression. In 9 patients
(patients 1 to 9) Southern analyses showed p16(INK4A) gene deletion or
rearrangement corresponding to the loss of 1 or both exons 1 and 2 detected by PCR analysis. In 1 patient (patient 10) a rearranged
p16(INK4A) gene was amplified by both the exon 1 and exon 2 primers. In
another patient (patient 11), a large deletion involving both
p16(INK4A) and p15(INK4B) genes was detected by Southern analysis, yet
p16 remained detectable by PCR analysis. Nonetheless, overall there was
excellent correspondence between the results given by the 2 techniques.
Southern blotting identified p16(INK4A) gene deletions in 10 patients
and gene rearrangements in 3 patients (Table 3). Among the 13 patients
in whom abnormalities were detected by PCR, neither exons 1 nor 2 of
p16 were detected in 11 patients (85%). The p15(INK4B) gene was
co-deleted with p16(INK4A) in 9 of 10 patients (data not shown).
Rearrangements of p15(INK4B) were not identified. In 1 case of a
p16(INK4A) deletion detected by Southern blotting (patient 1), exon 1 was absent but exon 2 was detected by PCR. Because the p15(INK4B) gene
also was detected, it is likely that a micro-deletion occurred in this
patient. In a second patient (patient 7) a p16(INK4A) gene
rearrangement was associated with loss of exon 2 but retention of exon
1 by PCR analysis.
Outcome analyses
We previously have found no association between occurrence of any
single gene abnormality (Rb or p53 only) and CR, overall, or
relapse-free survival.10 However, preliminary analyses
suggested a worse outcome for patients with aberrant expression of both Rb and p53.10 Therefore, we analyzed outcome data for the
patients in this study in relation to the number of genetic aberrations (or hits) in the cell cycle regulatory genes (Table
4). Patients with 0 to 1 abnormality in
cell cycle regulatory genes had a CR rate of 69%, whereas those with
more than 1 abnormality had a CR rate of 54% (not statistically
significantly different). However, the significance of these
comparisons is unknown because of the small sample size. Because 33 of
39 (85%) patients in this cohort have died (Figure
2), relapse-free survival provided no
additional information compared to overall survival. Patients with 0 to
1 abnormality in cell cycle regulatory genes had a median overall survival of 25 months (n = 26; 95% CI, 13 to 46 months) compared to
8 months (n = 13; 95% CI, 4 to 12 months) for those with 2 or 3 abnormalities (P < .01) (Figure 2).
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| Fig 2.
Overall survival of cohort patients
by number of cell cycle regulatory gene abnormalities.  0 to1
abnormality; ---2 to 3 abnormalities.
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Clinical (age, sex, race, marrow and peripheral blasts, WBC, peripheral
lymphocyte counts, hemoglobin, platelets) and laboratory (FAB
classification, blast lineage, myeloid antigen, and CD34 expression)
features did not differ significantly between these 2 patient groups
(data not shown). In addition, limited cytogenetic and molecular
genetic data available for these patients permitted an estimate of the
distribution of poor-risk cytogenetic characteristics between the 2 patient groups. Among the 26 patients with 0 to 1 regulatory gene
abnormality, 3 had both the Ph1 chromosome and BCR-ABL detected by
RT-PCR (2 subjects with p190 and 1 with p210); 1 had the p210 BCR-ABL
by RT-PCR; 2 had t4;11, 1 had t8;14, and 1 was
hypodiploid by cytogenetic analysis. Among the 13 patients with 2 or
more regulatory gene abnormalities, 1 had the p210 BCR-ABL fusion gene
by RT-PCR; 2 had t4;11, and 2 were hypodiploid by
cytogenetic analysis. These data suggest that poor-risk cytogenetics
was similarly distributed between the 2 groups defined by incidence of
cell cycle regulatory gene abnormalities. Therefore, taken together,
the similar clinical characteristics in the 2 groups indicate that
abnormalities of cell cycle regulatory genes may impact clinical outcomes.
Stem cell sorting in adult ALL
CD34 analyses and stem cell sorting were performed on a group of 16 adult patients with ALL registered on protocol S9400. The cellularity,
bone marrow blast count, frequency of CD34+ blasts, and p16(INK4A)
abnormalities are shown in Table 5. Bone
marrow cellularity ranged from 90% to 100%, and bone marrow blasts
ranged from 55% to 98%. The CD34+ blast frequency ranged from 1% to
96%, and 7 of 16 of these specimens had p16(INK4A) abnormalities by immunocytochemistry. In preliminary experiments, we confirmed that
p16(INK4A) deletions detected by immunocytochemistry were highly
concordant (more than 90%) with our Southern blotting and RT-PCR data
(Tsai T, unpublished observations).
The differentiation stage at which p16(INK4A) genetic hits were
acquired was investigated by sorting bone marrow or blood cells from de
novo adult patients with ALL using multivariate flow cytometry with
lineage markers CD19, CD33, CD34, and CD38. Three subpopulations were
discriminated: stem (CD34+lin), committed progenitor
(CD34+lin+), and (CD34lin+).
Representative flow cytometric analysis of a leukemic specimen (patient
15) is shown in Figure 1. A light scatter gate (R1) (Figure 1A) was
used to discriminate nucleated cells from debris. PI-positive cells
were excluded from the analyses (Figure 1B). CD34+lin (R5),
CD34+lin+ (R6) and CD34lin+ cells (R7) (Figure 1D) were
identified on the basis of immunofluorescence that exceeded the isotype
control (Figure 1C) and were sorted onto microscope slides.
CD34+lin cells, CD34+lin+, and CD34lin+ cells comprised between 0.01% to 6.33%, 0.06% to 61.92%, and 1.04% to 80.43%, respectively (data not shown).
p16(INK4A) expression in sorted cell subpopulations and unsorted
lymphoblasts from a subset of patients with or without p16 abnormalities was measured using immunocytochemistry (Table
6). p16 expression was present in the
CD34+lin compartment in 95% (median) of patients whose
lymphoblasts expressed p16(INK4A) (Table 6, Figure
3). In contrast, p16 expression was
observed in only 10% (median) of CD34+lin compartments from
patients with lymphoblasts displaying p16(INK4A) abnormalities.
Approximately 20% (median) of specimens from patients with
p16-aberrant lymphoblasts contained CD34+lin+ cells that expressed p16.
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| Fig 3.
Immunocytochemical determination of p16(INK4A) expression
in sorted bone marrow stem cells.
Adult ALL bone marrow was sorted as described in "Materials and
methods." Sorted stem cells (CD34+lin) were collected on
slides and stained for p16(INK4A) expression. The slide images were
captured using an inverted microscope at 125× magnification. The
images were transferred to a computer using a frame grabber board
(Target+; Truevision) and stored in computer files using Java image
analysis software (Jandel Scientific, Corte Madona, CA). Left panel:
sorted CD34+lin cells from a p16(INK4A)-positive ALL bone
marrow. Note positive nuclear localization. Right panel: sorted
CD34+lin cells from a p16(INK4A)-negative ALL bone marrow.
Nuclei are unstained.
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The frequency of p16-positive cells in CD34 subpopulations is shown in
Table 7. In 3 of 5 evaluable patients
(patients 11, 12, and 15), p16(INK4A) expression was effectively absent
in the CD34+lin compartment, whereas in 2 of 5 patients only
50% of cells expressed p16(INK4A). It is important to note that 3 of these patients (patients 11, 12, and 16) contained a low frequency (12% or less) of CD34+ lymphoblasts (Table 5), suggesting
that even in CD34 ALL, cells in the CD34+lin
compartment contained cell cycle regulatory gene abnormalities.
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Discussion |
Abnormalities of cell cycle regulatory genes are being reported with
increasing frequency in lymphoblasts of patients with adult ALL
(reviewed in references 4 and 25). However, to date there have been few
follow-up studies that systematically assessed the impact of these
abnormalities on clinical outcomes in groups of patients treated
uniformly. Tsai et al10 have examined influences of Rb and
p53 gene abnormalities on outcome measures of patients registered to
front-line and relapsed/refractory SWOG adult ALL protocols, and Stock
et al26 have performed similar analyses for p16(INK4A)
inactivation in adult patients enrolled in a front-line CALGB study.
These studies involved analyses 89 SWOG de novo ALL patients, 68 CALGB
de novo patients, and 26 refractory/relapsed patients (SWOG). In the
SWOG study, immunoblotting was used to assess expression of Rb and p53.
Loss of Rb expression occurred in 54 of 85 (64%) and p53
overexpression (indicative of p53 point mutations) in 16 of 75 (21%)
de novo adult ALL patients. No significant correlations of Rb or p53
abnormalities with outcome measures were identified. De novo adult ALL
patients with abnormalities of both Rb and p53 expression appeared to
have an increased rate of early death, but the number of patients in
this group was small.10 Likewise, adult patients with ALL
who had relapses had a higher frequency of p53 overexpression than de
novo patients (8 of 19 [42%] versus 16 of 75 [21%]), but the
difference was not statistically significant
(P = .09).10
In the CALGB study RT-PCR was used to detect p16(INK4A) mRNA, and
Southern analyses were used to detect deletions or genomic rearrangements of the p16(INK4A) gene.26 Deletions or
genomic rearrangements were detected in 19 of 68 (28%) of specimens.
In 49 patients with germline p16(INK4A), 13 (27%) lacked p16 mRNA. Overall, 46% of patients had inactivation of p16. Analyses of overall
survival and remission duration did not differ between patients with or
without p16 inactivation.26
We hypothesized that simultaneous occurrence of multiple tumor
suppressor gene abnormalities in adult ALL masks the prognostic effects
of any single gene abnormality.10 We examined the
relationship between multiple cell cycle gene abnormalities and outcome
in adult patients with ALL treated uniformly on a cooperative group protocol. Multiple aberrations of these genes were present in 13 of 39 patients (Table 2). These include Rb and p16 abnormalities (6 patients), p53 and p16 abnormalities (3 patients), Rb and p53 abnormalities (3 patients), and abnormalities of all 3 genes, Rb, p16,
and p53 (1 patient).
Sherr27 has described 2 major regulatory pathways of cell
cycling. In 1, the "Rb pathway," the CDK4 and
CDK6 inhibitory proteins, so-called INK4 proteins,
p16(INK4A), p15(INK4B), p18(INK4C), and p19(INK4D), regulate passage
through the G1/S transition. In the second pathway
p53-dependent checkpoints involving the p53, p21(WAF1/CIP1), p27(KIP1),
and p57(KIP2) proteins regulate G1/S transition. The
transcription activator, E2F, which binds Rb, is central to both
pathways. Hyperphosphorylation of Rb by CDKs releases E2F, thereby
enhancing transcription of target genes and cell cycling. The
cross-talk between these 2 pathways may compensate for single gene
abnormalities. For example, in situations with abnormal p16(INK4A), Rb
hyperphosphorylation can be blocked by the p53
pathway.28,29 Similarly, in patients with p53 alterations, the INK4 pathway would remain operative. Thus, defects of more than 1 cell cycle regulatory gene involved in both the INK4 and p53-dependent
pathways may be required to produce unregulated cell cycling and
thereby predispose subjects to worse clinical outcomes. Absence of Rb
protein, the most common defect documented in our study, would appear
to make any additional gene defects in either of these pathways
redundant. On that basis, it is not evident that acquisition of a
second "hit" by a lymphoblast already lacking Rb would confer any
cell cycling advantage. However, 6 of 9 patients had Rb abnormalities
with p16(INK4A) and 3 of 9 displayed both Rb and p53 abnormalities. One
patient had abnormalities of p16(INK4A), Rb, and p53. Hangaishi et
al30 reported 4 patients with lymphoid malignancies (230 specimens) with inactivations of both Rb and p16(INK4A). These authors
suggested that sequential acquisition of p16(INK4A) and Rb
abnormalities might have occurred in these patients. If p16
abnormalities were to precede Rb inactivations, subsequent acquisition
of Rb abnormalities could provide a growth advantage by abrogating
inhibitory cross-talk of other cyclin dependent kinase inhibitors
(CDKIs). This in turn would explain why patients with lymphoblasts
having 2 or more "hits" might do less well than those with only 1 "hit." We are unaware of studies in adult patients with ALL that
support or refute this hypothesis.
In our analysis, patients with 0 to 1 gene abnormality had a better
overall survival rate than those with more than 1 abnormality (25 months versus 8 months; P < .01) (Figure 2). Because these 2 patient groups did not differ with respect to other prognostic factors analyzed, these data support the hypothesis that multiple gene
abnormalities abolish cross-talk.
One potential limitation of this study is the small number of patients
with specimens suitable for analysis. With this as a consideration, it
is especially noteworthy that survival in the 2 groups defined by the
number of genetic hits differed significantly. Conversely, whereas the
difference in CR rates between these groups was not statistically
significant, this does not comprise strong evidence for the lack of
effect resulting from inadequate statistical power. A second potential
limitation is that our study group differed from the overall S8417
population by higher WBC, blast, and lymphocyte counts, features
associated with a poorer outcome in a previous analysis of this
study.5 Because cytogenetic analyses were not available for
all patients, we cannot exclude definitively the possibility that the 2 groups defined by regulatory gene abnormalities may have differed also
by poor-risk cytogenetic features. (Data that are available suggest
that this possibility may not be likely.) These factors may limit the
generalizability of the findings. However, these considerations provide
persuasive arguments for studying the hypothesis in a larger patient population.
Leukemias in which the neoplastic clone originates in a stem cell
compartment are likely to be more resistant to chemotherapy than those
originating at a later stage of differentiation. Stem cells tend to be
noncycling, express high MDR1, and are relatively radioresistant.31-33 One would further expect that this
resistance might influence CR rate, disease-free survival, or overall
survival. The phenotype CD34+lin has been used to define
hematopoietic stem cells.34-37 In 3 of 5 adult patients
with ALL with abnormal p16 expression in lymphoblasts, CD34+lin
cells also lacked p16(INK4A) expression, and in 2 of 5 patients only
50% of stem cells expressed p16(INK4A) (Table 7). In contrast, sorted
stem cells from patients with normal p16(INK4A) expression in
lymphoblasts also expressed p16(INK4A) (Table 6). Both stem cells and
lymphoblasts lacked p16(INK4A) expression in 3 of 5 patients. The
observation that more cells in the CD34+lin+ intermediate and the
CD34lin+ differentiated compartments expressed p16(INK4A), than
in the CD34+lin stem compartments suggests that the nonclonal,
nonneoplastic fraction of progenitor cells retains potential for normal differentiation.
The finding that p16(INK4A) silencing occurs in the stem (CD34+
lin) compartment strongly suggests that cell cycle gene
abnormalities are important etiologic factors in adult ALL
leukemogenesis. We do not know whether the other cell cycle regulatory
gene (Rb and p53) abnormalities also occur in the stem cell compartment
in adult ALL. However, the observation that p16(INK4A) alterations occur at this stage provides a potential biologic basis for
understanding how multiple gene abnormalities may contribute to
clinical disease and therapeutic resistance. Thus, elimination of the
CD34+lin stem cell subset harboring abnormalities of p16(INK4A)
or other cell cycle regulatory genes may be important to achieve
durable CRs in adult ALL.
| |
Acknowledgments |
We thank Cheryl Muzzi Adams for secretarial assistance and Sheryl
Dorsey, Sridevi Davalath, and Phyllis Eagan for technical assistance.
| |
Footnotes |
Submitted March 15, 1999; accepted November 26, 1999.
Supported in part by DHHS National Institutes of Health grants CA32102
and CA6032 to the SWOG Leukemia Biology and Cytogenetics Programs,
American Cancer Society Career Development Award CDA-96-85 (W.S.), and
American Cancer Society Clinical Fellowship Award 4497 (C.G.). M.L.S.
is a member of the City of Hope Cancer Center Program and is supported
by National Institutes of Health grants CA3333572 and CA30206.
Reprints: D. H. Boldt, Department of
Medicine/Division of Hematology, Mail Code 7880, University of Texas
Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX
78229-3900; e-mail: BOLDT{at}UTHSCSA.EDU.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction