Hematopathology / Outcome in Lenalidomide-Treated Multiple Myeloma
p53 Nuclear Expression Correlates With Hemizygous TP53
Deletion and Predicts an Adverse Outcome for Patients
With Relapsed/Refractory Multiple Myeloma Treated
With Lenalidomide
Mei-Hsi Chen, Connie X.Y. Qi, MD, Manujendra N. Saha, PhD, and Hong Chang, MD, PhD, FRCPC
CME/SAM
Key Words: Fluorescence in situ hybridization; FISH; Immunohistochemistry; del(17p13); p53; Lenalidomide
DOI: 10.1309/AJCPHC85DGAXZDBE
Upon completion of this activity you will be able to:
• identify the genetic risk factors in multiple myeloma.
• describe the correlation between p53 immunohistochemical analysis
and other genetic risk parameters in multiple myeloma.
• apply p53 immunohistochemical analysis as a potential surrogate
marker to predict an adverse clinical outcome in myeloma patients
treated with lenalidomide-based regimens.
Abstract
del(17p13)(TP53) seems to be an independent poor
prognostic factor in patients with relapsed/refractory
multiple myeloma (MM) receiving lenalidomide.
However, whether aberrant p53 nuclear expression
detected by immunohistochemical analysis can be
used as a surrogate marker for del(17p13)(TP53)
in prognostic evaluation of lenalidomide-treated
relapsed/refractory MM remains unclear. The
p53 expression in myeloma cells from 88 patients
was evaluated by immunohistochemical analysis,
and 17p13(TP53) gene status was examined by
fluorescence in situ hybridization (FISH). FISH
detected hemizygous del(17p13)(TP53) in 13 (15%),
and immunohistochemical analysis detected p53
nuclear expression in 11 cases (13%). del(17p13)
(TP53) and p53 expression were strongly correlated
(P < .0001). Furthermore, patients with aberrant
p53 nuclear expression had significantly shorter
progression-free and overall survival than patients
without this abnormality. Our results suggest that
p53 nuclear expression is associated with adverse
outcome in patients with relapsed/refractory MM
receiving lenalidomide-based therapy and that p53
immunohistochemical analysis may serve as a simple,
rapid method to predict del(17p13)(TP53) in this
patient subgroup.
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DOI: 10.1309/AJCPHC85DGAXZDBE
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Multiple myeloma (MM) is a plasma cell malignancy
characterized by heterogeneous biologic manifestations and
clinical course. MM remains largely fatal, and relapse occurs
frequently, even after high-dose therapy and autologous
stem cell transplantation. Lenalidomide is an analog of thalidomide, which exhibits immunomodulatory effects and
antitumor activity.1 The combination of lenalidomide plus
dexamethasone exhibited a higher overall response rate than
lenalidomide alone2 and has been shown to significantly
improve progression-free survival (PFS) and overall survival
(OS) in patients with relapsed MM.2,3
The influence of del(17p13)(TP53) on the clinical outcome of patients with MM treated with lenalidomide plus
dexamethasone has been investigated by several groups. In
patients with relapsed/refractory MM, the adverse prognostic
impact of del(17p13) was found to remain despite treatment
with lenalidomide plus dexamethasone4-6; thus, other therapeutic alternatives should be considered for patients with this
genetic abnormality. Moreover, in newly diagnosed cases of
patients receiving lenalidomide and dexamethasone as initial
therapy, del(17p13) was identified as one of the parameters
for high-risk MM, along with hypodiploidy, del(13q), t(4;14),
t(14;14), or a high plasma cell proliferative rate.7
Our group has previously reported that p53 immunohistochemical analysis correlates with hemizygous TP53
deletion and confers a poor prognosis in patients treated with
high-dose therapy and autologous stem cell transplantation.8
However, whether nuclear p53 expression has prognostic
significance in lenalidomide-treated relapsed/refractory MM
remains unclear. We therefore extended our investigation with
© American Society for Clinical Pathology
Hematopathology / Original Article
p53 immunohistochemical analysis in a cohort of patients
with relapsed/refractory MM receiving lenalidomide plus
dexamethasone.
Materials and Methods
Patients
A total of 88 patients with relapsed/refractory MM who
had received lenalidomide-based therapy were included in
this study. The inclusion criteria and treatment schedule were
as previously described.4 Briefly, lenalidomide was administered at 25 mg from days 1 to 21 on a 28-day schedule, with
dexamethasone given on days 1 through 4, 9 through 12, and
17 through 20 during the 4 initial cycles and on days 1 through
4 in subsequent cycles.2,3 The clinical and laboratory features
of the cases are summarized in ❚Table 1❚.
Fluorescence In Situ Hybridization
Clonal plasma cells in bone marrow aspirates archived
on cytocentrifuged slides were analyzed by interphase cytoplasmic fluorescence in situ hybridization (cIg-FISH) using
SpectrumGreen-labeled CEP17 and SpectrumOrange-labeled
17p13.1 (TP53) probes (Vysis, Downers Grove, IL). FISH
analyses for del(13q), t(4;14), and amp(1q21) were performed
as previously described.9,10 At least 200 plasma cells were
scored to determine the prevalence of each genetic abnormality. The positive cutoff level was established as exceeding 10%.
Immunohistochemical Analysis
Serial 5-μm sections of the bone marrow biopsy specimen were cut and deparaffinized. Antigen retrieval was
achieved by submerging the slides in 0.01 mol/L citrate
buffer (pH 6.0, 120°C) for 10 minutes. The myeloma cells
were immunostained for CD138 (Serotec, Oxford, England)
at a 1:100 dilution or p53 (DO-7, DAKO, Carpinteria, CA)
at a 1:200 dilution using a biotin-streptavidin-horseradish
peroxidase method (ABC kit, Vector Laboratories, Burlingame, CA). Slides were counterstained with hematoxylin.
Results were examined by 2 independent investigators
(M.-H.C. and C.X.Y.Q.) who were blinded to the patient’s
del(17p13) status and clinical outcome. Cases in which p53
stained 10% or more of the CD138+ myeloma cell nuclei
were considered positive.
Statistical Analysis
Categorical data were analyzed by using the Fisher exact
test or the χ2 test, and continuous variables were compared
by using the Mann-Whitney test. The Kaplan-Meier method
was used to calculate survival probability. PFS was calculated
from the start of lenalidomide therapy to the date of progression, death, or last follow-up; OS was calculated from the start
of therapy to death or last follow-up. The log-rank test was
used to analyze the difference between survival curves. A P
value less than .05 was considered of statistical significance.
Statistical analysis was performed using SPSS, version 16.0
(SPSS, Chicago, IL).
❚Table 1❚
Clinical Features According to p53 Expression Status*
Immunohistochemical Result for p53
Clinical Feature
Total (n = 88)
Positive (n = 11)
Negative (n = 77)
P
Sex (M/F)
Age (y)
Hemoglobin concentration (g/L)
WBC count (× 109/L)
Platelet count (× 109/L)
Calcium (mmol/L)
β2-Microglobulin (mg/L)
Creatinine (μmol/L)
Albumin (g/L)
Prior therapies, No. (%)
≥3
Thalidomide
Bortezomib
Autologous stem cell transplantation
Response to lenalidomide plus dexamethasone, No. (%)
Responsive†
Nonresponsive‡
53/35
56 (30-75)
106 (76-147)
5.4 (1.6-16.7)
218 (44-457)
2.28 (1.98-3.75)
2.40 (0-20.65)
78 (32-443)
39 (26-54)
8/3
54 (45-75)
117 (93-141)
5.5 (3.7-8.9)
210 (64-400)
2.31 (2.00-2.55)
1.96 (1.31-6.95)
72 (39-118)
41 (32-43)
45/32
57 (30-75)
106 (76-147)
5.4 (1.6-16.7)
219 (44-457)
2.26 (1.98-3.75)
2.75 (0-20.65)
81 (32-443)
39 (26-54)
.515
.126
.098
.437
.398
.598
.114
.354
.054
43 (49)
35 (40)
58 (66)
74 (84)
6 (55)
5 (45)
7 (64)
8 (73)
37 (48)
30 (39)
51 (66)
66 (86)
.990
.866
.682
.537
.147
65 (74)
23 (26)
6 (55)
5 (45)
59 (77)
18 (23)
*
Data are given as median (range) unless otherwise indicated. Laboratory values are given in Système International units; conversions to conventional units are as follows:
albumin (g/dL), divide by 10; calcium (mg/dL), divide by 0.25; creatinine (mg/dL), divide by 88.4; hemoglobin (g/dL), divide by 10; β2-microglobulin (mg/L), divide by 1.0;
platelet count (× 103/μL), divide by 1.0; and WBC count (/μL), divide by 0.001.
† Includes complete response, near complete response, and partial response.
‡ Includes minimal response, stable disease, and progressive disease.
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Results
Immunohistochemical Studies
Myeloma cells in all 88 cases studied stained positively
for CD138, a plasma cell marker, but only 11 (13%) were
positive for p53 nuclear expression by immunohistochemical
analysis. Among the 11 p53 immunohistochemically positive
cases, the staining intensity ranged from moderate to strong and
the median percentage of p53-stained myeloma cells was 40%
(range, 10%-90%). Myeloma cells in p53 immunohistochemically negative cases were mostly p53 nonimmunoreactive, with
a few cases having rare (<2%) p53-immunostained nuclei.
Correlation of Immunohistochemical Findings With FISH
All 88 cases had bone marrow aspirates available for
retrospective molecular cytogenetic analysis by FISH. In 23
(27%) of 86 cases, there was a 13q deletion; 12 (15%) of
82 had t(4:14); and 32 (41%) of 78 had amp(1q21). Of the
88 relapsed/refractory MM cases, 13 (15%) contained hemizygous 17p13 deletion shown by FISH. None of the cases
tested was positive for homozygous 17p13 deletion. Of the
13 del(17p13)+ cases, 11 (85%) expressed p53 detected by
immunohistochemical analysis, whereas all (100%) of the 11
immunohistochemically p53+ cases had del(17p13) shown by
FISH. del(17p) and p53 expression were strongly correlated
(P < .0001) ❚Image 1❚. There was no statistically significant
association between p53 nuclear expression and other genetic
abnormalities, including del(13q), t(4;14), or amp(1q21)
❚Table 2❚.
Correlation With Clinical Outcomes
All 88 patients with lenalidomide-treated MM had evaluable clinical responses. The overall response rate to lenalidomide plus dexamethasone in this cohort was 74% (65/88),
including 4 (5%) with complete response, 12 (14%) with
near-complete response, 15 (17%) with very good partial
response, and 34 (39%) with partial response. In 8 cases (9%),
there was minimal response, 5 (6%) cases showed stable disease, and in 10 (11%), disease was progressive. There was no
significant difference in response rate between patients with
or without p53 nuclear expression. No significant correlation
❚Table 2❚
Correlation of p53 Expression and Genetic Abnormalities*
Immunohistochemical
Result for p53
Genetic
Abnormality
del(13q)
Positive
Negative
del(17p)
Positive
Negative
t(4;14)
Positive
Negative
amp(1q21)
Positive
Negative
*
All
(n = 88)
Positive
(n = 11)
Negative
(n = 77)
23/86 (27)
63/86 (73)
6 (55)
5 (45)
17/75 (23)
58/75 (77)
13 (15)
75 (85)
11 (100)
0 (0)
2 (3)
75 (97)
12/82 (15)
70/82 (85)
2 (18)
9 (82)
10/71 (14)
61/71 (86)
32/78 (41)
46/78 (59)
6 (55)
5 (45)
26/67 (39)
41/67 (61)
P
.06
<.0001
.660
.344
Data are given as number (percentage) or as number/total (percentage).
B
A
❚Image 1❚ A, Expression of p53 by immunohistochemical analysis in a bone marrow biopsy specimen from a patient with
multiple myeloma (×1,000). B, The myeloma cell (upper left) of the bone marrow aspirate from the same patient showed TP53
deletion (1 red signal) by interphase cytoplasmic fluorescence in situ hybridization. cIg, cytoplasmic immunoglobulin; CEP,
centromere probe.
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© American Society for Clinical Pathology
Hematopathology / Original Article
was observed between p53 nuclear expression and sex, age, or
hemoglobin, calcium, β2-microglobulin, creatinine, albumin,
or C-reactive protein levels (Table 1). None of these clinical
parameters was found to impose a significant influence on
clinical response (data not shown).
The median PFS and OS for this cohort were 9.4 months
(95% confidence interval, 5.3-13.4 months) and 28.2 months
(95% confidence interval, 20.3-36.2 months), respectively.
Patients with p53 nuclear expression had a significantly
reduced PFS (3.4 vs 11.0 months; P = .021) and OS (7.2
vs 28.8 months; P = .018) compared with patients without
p53 expression ❚Figure 1❚. The PFS and OS for del(17p13)+
patients were also significantly shorter (3.4 vs 11.1 months;
P = .005; 12.1 vs 28.8 months; P = .028). None of the other
clinical features or genetic abnormalities significantly influenced the survival outcome of this cohort (data not shown).
Discussion
Novel therapies have changed the landscape of MM
treatment and provided survival advantages for patients with
relapsed/refractory MM.11 Unlike t(4;14), del(17p13) seems
to be an adverse genetic abnormality that cannot be overcome
by lenalidomide-based treatment.4-6 It would therefore be of
great clinical value if a simple, robust assay such as immunohistochemical analysis could be used to predict this poor
risk factor, especially for centers where FISH is not available. Herein, we report, for the first time, that p53 nuclear
B
1.0
1.0
0.8
0.8
Survival Probability
Survival Probability
A
expression in immunohistochemical analysis as a surrogate
marker for del(17p13) predicted an inferior clinical outcome
in patients with relapsed/refractory MM receiving lenalidomide plus dexamethasone.
We detected aberrant p53 nuclear expression in 11 (13%)
and hemizygous 17p13 deletion in 13 (15%) of 88 cases
studied. Of the 88 patients, 75 (85%) had neither del(17p13)
nor aberrant p53 nuclear expression, as detected by FISH and
immunohistochemical analysis, respectively. All 11 cases with
nuclear p53 expression were positive for 17p deletion. Thus,
by using nuclear p53 expression to predict 17p deletion, the
positive predictive value is 100% and the negative predictive
value is 97%; the sensitivity is 85% and the specificity is 100%.
p53 immunohistochemical analysis relies on the increased
stability of p53 protein for detection,12 and aberrant p53 expression in human cancer is readily detectable by this method as
TP53 is commonly mutated in the tumor cells.12-14 Although
mutational analysis was not performed in this study, it is possible that mutation of the undeleted allele has occurred in many
of the hemizygous del(17p13) cases because TP53 mutation
was found to be strongly associated with del(17p13).15,16
Moreover, other epigenetic mechanisms resulting in p53 overexpression/stabilization such as positive regulation of p53 via
up-regulation of p14(ARF)17 or microRNA-mediated downregulation of MDM218 could also have contributed to aberrant p53 expression. However, there were 2 cases positive for
hemizygous del(17p13) but negative for p53 immunostaining
in our cohort. The reason for such discordance is unclear. It is
0.6
0.4
p53–
0.6
p53–
0.4
p53+
0.2
0.2
p53+
0.0
0.0
0
20
40
60
80
Progression-Free Survival Time (mo)
100
0
20
40
60
80
100
Overall Survival Time (mo)
❚Figure 1❚ A, Progression-free survival according to p53 nuclear expression as detected by immunohistochemical analysis (P =
.021; log-rank test). B, Overall survival according to p53 nuclear expression as detected by immunohistochemical analysis (P =
.018; log-rank test). In A and B, p53 nuclear expression was negative in 77 cases and positive in 11.
© American Society for Clinical Pathology
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unlikely due to immunohistochemical technical issues because
repeated staining for these 2 samples yielded the same result.
It is probable that the remaining allele in these 2 cases retained
a wild-type because not all hemizygous del(17p13) cases have
the TP53 mutation.16 Alternatively, these cases might have a
mutated TP53 gene that leads to deletion or truncation of the
protein, which does not accumulate, thus, could not be detected
by immunohistochemical analysis.12,19
Recently, 2 groups have studied p53 immunohistochemically in patients with MM treated with other novel therapeutic
agents. Kelley and colleagues20 reported that p53 immunoreactivity was linked to significantly reduced PFS and OS in
newly diagnosed patients undergoing thalidomide therapy,
but not in relapsed/refractory MM. Dawson and coworkers21
found that cytoplasmic p53 immunohistochemical expression
was associated with poor response to bortezomib in relapsed
MM, but did not adversely influence patient survival in their
cohort. However, FISH was not performed, and the correlation
between p53 immunohistochemical results and del(17p13)
status was not addressed in these 2 studies.20,21 Nevertheless, these reports, together with our current analysis, suggest
the potential clinical relevance of p53 immunohistochemical
analysis in the era of novel therapies for MM. The value of p53
immunohistochemical analysis should be further confirmed in
larger, prospective clinical trials with novel agents for MM.
We have demonstrated that nuclear p53 expression
accurately predicts hemizygous TP53 deletion and adverse
outcome in patients with relapsed/refractory MM receiving
lenalidomide-based therapy. Because immunohistochemical
analysis is a widely available, rapid, and inexpensive laboratory technique, p53 immunohistochemical analysis can readily be adopted in the clinical setting to identify this high-risk
subset of patients with MM for alternative therapy.
From the Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Canada; and Department
of Laboratory Hematology, Laboratory Medicine Program,
University Health Network, Toronto.
Supported in part by grants to Dr Chang from the Canadian
Institute of Health Research, Ottawa, and the Leukemia &
Lymphoma Society of Canada, Toronto.
Address reprint requests to Dr Chang: Dept of Laboratory
Hematology, University Health Network, 200 Elizabeth St,
11E-413, Toronto, ON, M5G 2C4.
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© American Society for Clinical Pathology