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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4342-4349
By
From the Dipartimento di Biotecnologie Cellulari ed Ematologia,
Università "La Sapienza," Rome; Dipartimento di Scienze
Biomediche ed Oncologia Umana, Università di Torino,
Torino; and Laboratorio di Oncogenesi Molecolare, Istituto
Regina Elena, Rome, Italy.
We have analyzed by immunocytochemistry (ICC) the frequency of p53
protein expression in 181 cases of B-cell chronic lymphocytic leukemia
(CLL) followed at a single institution to assess the relationship
between p53 and the clinical and morphological features of the disease,
as well as the possible involvement of this protein in the pathogenesis
of the more aggressive forms of CLL. The overall frequency of p53
protein positivity in CLL was 15% (27 of 181 cases). There were no
significant differences in age, sex, absolute lymphocyte count, or
lymphocyte doubling time between p53-positive and -negative patients.
By contrast, p53-positive patients had a significantly higher
percentage of prolymphocytes (P = .002) and a significantly
lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive
cells and the percentage of cells in cycle assessed by the monoclonal
antibody Ki-67. When the percentage of p53 positivity was correlated
with the clinical stage of the disease, the proportion of p53-positive
cases increased significantly from Binet's stage A (8 of 108; 7.4%),
to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease,
showing a low expression at diagnosis (8 of 112; 7.1%) and a
significantly higher expression in patients studied during the course
of the disease (7 of 35; 20%) and, to a further extent, with disease
progression (12 of 34; 35.3%) (P = .0001). The association
of p53 protein expression with mutations in the gene was confirmed by
direct sequence of the entire cDNA in 15 of the 17 ICC positive cases
tested (88%). A significantly shorter treatment-free interval from
diagnosis (P = .003) and a poorer response to therapy
(P = .007) was observed in p53-positive compared with
p53-negative patients. Overall survival from the time of diagnosis, as
well as from the time of p53 protein analysis, was significantly
shorter in patients with p53 protein expression (P = .03 and
.0001, respectively). Moreover, in multivariate analysis, p53
expression and stage C were independently associated with a short
survival. The results of this study indicate that in CLL the expression
of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10%
prolymphocytes), advanced clinical stage, progressive
disease, poor response to therapy, and short survival.
B-CELL CHRONIC LYMPHOCYTIC leukemia (CLL)
is the most common leukemia in the Western world. It is characterized
by a highly variable clinical course, with some patients living several
years untreated without changes in their clinical status and others showing a more rapid disease progression and a significantly shorter survival.1 The biological mechanisms underlying such
variability in clinical behavior remain largely unclear. The issue of
identifying in CLL parameters, which bear predictive implications, is
becoming of greater relevance in view of the progressive change in the management of a disease for which, until recently, observation and
conservative treatment was the strategy of choice for the majority of
patients. Several considerations have contributed to this modified
attitude, including the knowledge that about 20% of patients are
diagnosed with CLL at 55 years or younger, that 60% to
70% of patients at the time of diagnosis have an early stage disease,
that the biological age of patients in their 60s has dramatically
improved, that the overall life expectancy is progressively increasing,
and that we now have a broader therapeutic armamentarium for patients
with CLL.2
The p53 tumor suppressor gene, located on chromosome 17 band p13.1, is
a transcription factor that is involved in the cell cycle arrest and
induction of apoptosis in genetically damaged cells. Mutations or
deletions of the p53 gene may facilitate the transmission of a genetic
damage and the emergence of neoplastic clones with a survival
advantage.3,4 p53 is the most frequently altered gene in
human cancer, being mutated in approximately 50% of all human
tumors.5 This gene is known to be altered in a number of
hematologic malignancies, but the frequency of p53 gene mutations tends
to be low in most lymphoid malignancies and is found mainly in
aggressive non-Hodgkin's lymphoma (NHL),6-11 progressive CLL,12-18 and B-cell chronic prolymphocytic leukemia
(PLL).19 Cases of indolent lymphoma and most cases of CLL
have so far been reported to be negative for p53 mutations.
The p53 gene encodes a p53-kD phosphoprotein that is normally present
in the nucleus of the cells. The wild-type p53 protein has a short
half-life and cannot be detected in the cell nucleus of most normal
human tissues. In contrast, mutated p53 has a prolonged half-life and
becomes detectable by immunologic techniques using anti-p53 monoclonal
antibodies (MoAb).20,21 For several years the immunologic
identification of the p53 protein in human tumors has been considered a
marker of p53 gene mutation.3,21 However, more recent
studies in high grade NHL, CLL, and PLL have shown that p53 expression
may not be associated with detectable gene mutations, whereas a mutant
p53 gene can have an undetectable protein,8,19,22-29
indicating that gene mutation and protein detection may not be
associated.
In the present study, we have analyzed by immunocytochemistry (ICC) the
frequency of p53 protein expression in 181 CLL patients followed at a
single institution to assess the relationship between p53 and the
clinical and morphological features of the disease, the possible
involvement of this protein in the pathogenesis of the more aggressive
forms of CLL, and its impact on the response to treatment and overall
survival. To evaluate whether p53 positivity to ICC was due to
mutations in the p53 gene or to other mechanisms of p53 stabilization,
a direct sequence of the entire protein coding region was performed in
the majority of ICC-positive cases. p53 expression increases in
association with cell proliferation.30,31 A relationship
between proliferation and p53 expression has been reported in
NHL11,32 in which the degree of p53 expression correlated
with prognosis, histological grade, and resistance to treatment.
Because a correlation between the percentage of leukemic cells in cycle
and the stage and clinical behavior of CLL has been
documented,33 we have also investigated a possible
relationship between p53 expression and positivity with Ki-67, a MoAb
that recognizes a nuclear antigen expressed during most phases of the
cell cycle.
Patients.
Peripheral blood samples from 181 cases of CLL referred to our
institution were studied. Informed consent was obtained from all
patients. Diagnosis, clinical staging, and response were based on the
criteria recommended by the International Workshop on
CLL.34 Cases were classified as CLL (n = 147) or CLL with
greater than 10% prolymphocytes (CLL/PL) (n = 34) on the basis of
May-Grunwald Giemsa-stained peripheral blood films.35 All
samples that entered the study were CD19+,
CD20+, CD5+, and CD23+. B-cell
clonality was established using anti- ICC.
Mononuclear cells were isolated from heparinized peripheral blood by
Lymphoprep density gradient centrifugation (Nycomed Pharma AS, Oslo,
Norway). Cytospins were prepared with a concentration of 5 × 104 cells per slide, air dried overnight,
wrapped in aluminum foil, and stored at DNA sequencing.
Total RNA was isolated from frozen pellets of mononuclear blood cells
using the RNeasy mini kit (Qiagen, Hilden, Germany) following the
manufacturer's instructions. Reverse transcription was performed on 1 to 2 µg of total RNA using 200 U/sample of Moloney murine leukemia
virus reverse transcriptase (Pharmacia Biotech AB, Milan, Italy) and
random primers. Each cDNA preparation was amplified with 4 U of Taq
polymerase (Ampli Taq; Perkin-Elmer AB, Milan, Italy) according to the
manufacturer's instructions, using a Perkin Elmer 9600 PCR equipment
programmed to perform 38 cycles. DNA sequencing primers were
synthesized according to the cDNA sequence of p53 messenger RNA.
Polymerase chain reaction (PCR) primers were prepared by Pharmacia
Biotech AB. Four sets of primers were used to cover the complete
protein coding region of the p53 cDNA.39 Sequencing
reactions were performed as described39 using
streptavidin-coupled Sepharose HP attached to the teeth of
plastic combs (solid-phase sequencing combs). The combs were removed
from the sequencing reaction mixtures and inserted into the wells of an
automatic laser fluorescence (ALF) DNA sequencer (Pharmacia Biotech,
Uppsala, Sweden). After 10 minutes, the comb was carefully removed from
the gel apparatus and electrophoresis initiated. Evaluation of the p53
sequences was performed with the aid of the DNAstar (DNAStar Inc,
London, England) software program.
Statistical analysis.
Two-sided p53 expression in CLL.
With the immunocytochemical method used to identify p53-positive cells,
intense brown nuclear staining of the positive population was obtained,
with good preservation of morphological details (Fig 1A). The reaction was always confined
to the nucleus. Cytospins with a mixture of Raji cells (p53 positive)
and normal peripheral blood lymphocytes (p53 negative) were used as
controls (Fig 1B). Because no p53-positive cells were observed in the
10 normal peripheral blood samples used as negative controls, CLL was
considered positive when at least 1% of lymphoid cells showed a strong
nuclear staining with the anti-p53 MoAb.
Patients' characteristics according to p53 immunostaining.
Based on the immunostaining pattern, the 181 patients were subdivided
into two groups: (1) p53-negative (n = 154) and (2) p53-positive (n = 27) CLL. There were no significant differences in age, sex, absolute
lymphocyte count, or LDT between the two groups. By contrast,
p53-positive cases had a significantly higher percentage of
prolymphocytes and a significantly lower percentage of residual
CD3-positive T lymphocytes (Table 2).
p53 sequence.
Among the 27 cases positive for p53 staining by ICC, 17 were studied to
verify whether positive ICC was due to gene mutations or to other
mechanisms of p53 stabilization. Total RNA was extracted from blood
mononuclear cells, reverse transcribed, and the entire protein coding
region sequenced. p53 mutations were found in 15 cases (88%)
(Table 3). Missense mutations were identified in all 15 samples. One out of frame mutation was found in patient no. 4 together
with other two missense mutations. No nonsense mutations or insertions
were found. The most frequent mutation, found in 10 cases, was a C
Frequency of Ki-67 expression in p53-positive CLL.
With the immunocytochemical method used to identify
Ki-67-positive cells, intense brown nuclear staining
of the positive population was obtained, with good preservation of
morphologic details. The results of immunostaining are summarized in
Table 1. The percentage of Ki-67 positivity in the p53-positive cases
was 4.7% ± 1.2%. When the percentage of Ki-67-positive cells was
correlated with the morphology, a significantly higher percentage of
cells in cycle was found in CLL/PL (8.8% ± 2.5%) compared with
CLL (2.0% ± 0.4%) cases (P = .004). There was no
correlation between the percentage of p53- and Ki-67-positive
cells.
Response to therapy and survival.
Of the 154 p53-negative patients by ICC, 82 (53%) have so far never
required treatment, 10 (7%) were lost to follow-up, and 62 (40%) were
treated with chlorambucil plus prednisone (n = 42), fludarabine plus
prednisone (n = 18), or CHOP (n = 2) as first line therapy. Nineteen of
the 27 p53-positive patients (70%) were treated with chlorambucil plus
prednisone (n = 16), fludarabine and prednisone (n = 2), and CHOP (n = 1). A significantly poorer response to therapy was observed in the
p53-positive patients (P = .007)
(Table 4). A similar poor response to
therapy was observed in the p53-positive group when the analysis was
focused on the 112 patients studied at diagnosis (data not shown). The same difference in response rate was observed between the 16 (10%) p53-negative and nine (33%) p53-positive patients who
required treatment within 3 months from diagnosis (no response: 31%
v 78%, respectively; P = .04).
Structural alterations and point mutations of the p53 tumor suppressor
gene have been shown in 10% to 15% of CLL; they have been associated
with poor survival and nonresponse to therapy and reported in rare
cases of high-grade lymphoma evolved from CLL (Richter's
transformation), suggesting that p53 may play a role in the clinical
course of the disease and in the transformation of some cases of
CLL.12-18 Less attention has been paid to the significance
of p53 protein expression in CLL, although an association with poor
survival and nonresponse to therapy has been observed in small series
of CLL.21,40 p53 expression has been shown to be a fairly
common feature in high-grade NHL, significantly associated with a short
survival and not always secondary to p53 gene mutation. Several studies
have, in fact, shown that in some high grade NHL the occurrence of
positive immunostaining does not reflect point mutations in the p53
gene and vice versa.8,11,22-24,26-28,41-44 Therefore, it is
evident that the relationship between p53 protein detection and the
existence of gene mutations is more complex than initially expected and
that other mechanisms of p53 stabilization are frequently operating in
NHL.27
Submitted August 13, 1997;
accepted January 29, 1998.
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Abstract
Introduction
Abstract
and anti-
immunoglobulin (Ig) light chain reagents. Due to the weak surface Ig expression, staining of fixed cells by immunoperoxidase was necessary in most cases
to show the presence of monoclonal Ig light chains. One hundred eight
patients were men and 73 were women. The mean age was 66 ± 10 years. According to Binet's staging system, 108 patients were in stage
A, 49 in stage B, and 24 in stage C. Within CLL stage A, two groups
were considered A' (Hb>12 g/dL and lymphocytes < 30 × 109/L) (n = 74) and A" (Hb <12 g/dL and/or
lymphocytes > 30 × 109/L) (n = 34).
20°C until
immunostaining. Because we have observed a progressive decrease in p53
protein expression within a few months from the cytospin preparations,
all cases were studied in the 2 months subsequent to storage at
2 or Student's t-test were used to
compare age, sex, absolute lymphocyte count, CLL/PL morphology,
lymphocyte doubling time (LDT), percentage of Ki-67-positive and
CD3-positive lymphocytes, stage and phase of the disease, TFI, and
response to therapy between patients with and without p53 expression.
The Kruskal-Wallis test was used to compare the percentage of
p53-positive cells in the three groups of patients subdivided according
to the clinical stage and phase of the disease. Survival was defined as
the time from diagnosis or from p53 analysis to death or to the last
observation. Comparison between curves was performed by the two-sided
log-rank test and actuarial curves were constructed according to the
Kaplan-Meier method. Survival multivariate analysis was performed
according to Cox's proportional hazard model.
) and p53-negative (
)
patients subdivided according to the clinical stage.
T transition at codon 143 (exon 5), which resulted in a
substitution of a valine with an alanine. More than one mutation was found in eight
samples. Moreover, four of the 14 different mutations found were
located outside the evolutionary conserved regions of the p53 coding
for the subdomains I-V. The percentage of p53-positive cells
stained by ICC was high in the majority of cases studied by cDNA direct
sequence; however, p53 mutations were detected also in four cases (no.
9, 12, 13, and 14), which had a low percentage of p53-positive cells.
