Immunohistochemically Detected Expression of 3 Major Genes (CDKN2A/p16, TP53, and SMAD4/DPC4) Strongly Predicts Survival in Patients With Resectable Pancreatic Cancer
Immunohistochemically Detected Expression of 3 Major Genes (CDKN2A/p16, TP53, and SMAD4/DPC4) Strongly Predicts Survival in Patients With Resectable Pancreatic Cancer
Immunohistochemically Detected Expression of 3 Major Genes (CDKN2A/p16, TP53, and SMAD4/DPC4) Strongly Predicts Survival in Patients With Resectable Pancreatic Cancer
Copyright © 2013 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Annals of Surgery r Volume 258, Number 2, August 2013 p16, p53 and Smad4 in Pancreatic Cancer
genetic status of these 4 genes and clinicopathological features, in- (>10 metastases, often hundreds to thousands) in 1 or more organ
cluding survival, have been analyzed, most of the previous reports sites, as previously reported.30
had focused on the relationships between the status of individual
genes and outcome, and they have been inconsistent and frequently Immunohistochemistry
conflicting.11 Furthermore, although genetically engineered mouse Paraffin-embedded samples of the primary carcinomas from
models have been providing the information that the concomitant 106 patients were immunostained for p16, p53, and Smad4/Dpc4.
expression of these mutated genes is essential to invasion and metas- At least 3 different slides were stained for each case to evaluate het-
tasis progress in PDACs,12–14 the influence of the coexistence of erogeneity within the primary cancer. Immunohistochemical label-
these gene alterations in the same PDAC on biological behavior and ing was carried out using a Bond Max instrument (Leica Microsys-
survival outcome is also conflicting.15,16 tems, Wetzlar, Germany), as previously described.31 An antihuman
The objective of the current study was therefore to clar- p16 monoclonal antibody (clone E6H4, ready-to-use; Roche mtm
ify the clinical implications of the status of these “mountain” laboratories AG, Heidelberg, Germany), an antihuman p53 mouse
genes and their combinations in PDAC. Fortunately, it has been monoclonal antibody (clone DO-7, ready-to-use; DakoCytomation,
reported that immunohistochemical labeling of p16, p53, and Grustrup, Denmark), and an antihuman Smad4/Dpc4 mouse mono-
Smad4/Dpc4 reflects the genetic status of CDKN2A/p16,17 TP53,18-24 clonal antibody (clone B-8, diluted 1:100; Santa Cruz Biotechnol-
and SMAD4/DPC4,25,26 respectively. Therefore, we investigated ogy, Santa Cruz, CA) were used. Islet cells in each case served as
immunohistochemically the status of these 3 “mountain” genes an internal control for positive p16 immunolabeling. Immunohisto-
(CDKN2A/p16, TP53, and SMAD4/DPC4) among the total of 4 genes chemical labeling of p16 was scored as intact (positive), indicating
because the KRAS gene is mutated in virtually all PDACs,7,15,27 and the presence of an intact gene, or lost (negative), indicating a deletion
we analyzed the relationships of their status, alone and in combination, or inactivating mutation of the gene had occurred (Fig. 1).17,32 For
with clinicopathological findings in patients with PDAC undergoing p53 immunolabeling, scattered acinar and ductal cells with nuclear
surgical resection.
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Oshima et al Annals of Surgery r Volume 258, Number 2, August 2013
labeling were typically present in the adjacent normal tissue ber 2011. The median survival was estimated using the Kaplan-Meier
(Fig. 2A). Therefore, p53 immunolabeling was considered to be “ab- method, and the difference was tested using the log-rank test. The 1-,
normal” when either the neoplastic cells showed a virtual absence 3- and 5-year survival rates were estimated using life tables. P values
of immunolabeling compared with adjacent normal tissue (immuno- less than 0.05 were considered statistically significant. Variables that
labeling in <5% of neoplastic cells), suggesting the presence of an were found to be significant on univariate analysis at P < 0.1 were
intragenic deletion, nonsense or frameshift mutation (Fig. 2D), or included in multivariate analysis in a backward stepwise fashion. Cox
showed robust nuclear accumulation of immunolabeled protein in proportional hazards models were generated for multivariate analy-
≥30% of neoplastic cells compared with adjacent normal cells (Fig. sis. Statistical analysis was performed using IBM SPSS Statistics 20
2C).18-24 Normal acinar cells, ductal cells, islet cells, and stromal cells (IBM, Armonk, NY).
in each case served as internal controls for positive Smad4/Dpc4 im-
munolabeling. Immunohistochemical labeling of Smad4/Dpc4 was
scored as intact (positive), indicating the presence of an intact gene, RESULTS
or lost (negative), indicating a deletion or inactivating mutation of the
gene had occurred (Fig. 3).25 Negative controls for each of the anti- Clinicopathological Characteristics and Outcome
bodies were included using nonimmune serum instead of the primary The cohort of 106 patients (Table 1) consisted of 44 women and
antibodies. 62 men. The mean age at operation was 68.0 years, with a median age
of 69.5 years and range of 36–86 years. Thirty-six (34.0%) patients
were alive at the census date (December 2011). The 30-day mortality
Statistics rate was 0%. Sixty-six (62.3%) patients died of PDAC and 4 (3.8%) of
Frequency distributions were compared by χ 2 test. Continuous other causes. The median overall survival was 22.1 months, with 1-,
variables were compared using the Student t-test. The principal out- 3-, and 5-year survival rates of 71.9%, 28.8%, and 17.5%, respec-
come measure was length of survival as measured from the time of tively, in all 106 patients. The majority of tumors were well dif-
the original surgery. Patients alive at the time of follow-up point were ferentiated (57.5%), followed by moderately differentiated (28.3%),
censored. The last follow-up period for patients still alive was Decem- and 14.2% of tumors were poorly differentiated. Most tumors were
FIGURE 2. Typical immunohistochemical labeling profiles of p53 in pancreatic tissues. A, Normal pancreatic tissue. Scattered
ductal and acinar cells with positive nuclear labeling are present. B, Pancreatic ductal adenocarcinoma (PDAC) showing a
“normal” pattern of p53 immunohistochemical labeling. Scattered cells in the neoplastic glands with positive nuclear labeling are
present. C, Example of PDAC with diffusely positive nuclear labeling for p53. D, Example of PDAC with loss of nuclear labeling
for p53 (right side). In contrast, scattered reactive pancreatic ducts (N) with positive nuclear labeling are present in the adjacent
nonneoplastic tissue (left side).
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Annals of Surgery r Volume 258, Number 2, August 2013 p16, p53 and Smad4 in Pancreatic Cancer
Immunohistochemical Analysis
Immunohistochemical results are also presented in Table 1.
Loss of p16 immunolabeling was identified in 71 (67.0%) of the 106
patients (Fig. 1). Abnormal immunolabeling of p53 was detected in
86 (81.1%) of the 106 PDACs (Fig. 2). Twenty-seven (25.5%) PDACs
showed a virtual absence of p53 immunolabeling compared with ad-
jacent normal tissue (immunolabeling in <5% of neoplastic cells)
(Fig. 2D) and 58 (54.7%) showed robust nuclear accumulation of im-
munolabeled p53 protein in ≥30% of neoplastic cells compared with
adjacent normal cells (Fig. 2C). Intratumoral heterogeneity of p53 im-
munolabeling was observed in 1 (1.0%) PDAC. Loss of Smad4/Dpc4
immunolabeling was identified in 58 (54.7%) of the 106 PDACs
(Fig. 3) and intratumoral heterogeneity in 6 (5.7%). Immunohis-
tochemically detected heterogeneity of p53 and Smad4/Dpc4 was
categorized into abnormal immunolabeling of p53 and loss of im-
munolabeling of Smad4/Dpc4, respectively.
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Oshima et al Annals of Surgery r Volume 258, Number 2, August 2013
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Annals of Surgery r Volume 258, Number 2, August 2013 p16, p53 and Smad4 in Pancreatic Cancer
TABLE 1. (Continued)
Overall Survival Disease-Free Survival
Variable No. of Patients (%) Median (Months) Log-Rank (P Value) Median (Months) Log-Rank (P Value)
Locoregional recurrence
Present 56 (52.8)
Absent 45 (42.5)
Unknown 5 (4.7)
First sign of recurrence
No recurrence 23 (21.7)
Locoregional 27 (25.5) 24.8 0.004 11.0 0.047
Distant metastases 38 (35.8) 20.1 4.0
Synchronous 17 (16.3) 9.3 4.9
Unknown 1 (0.9)
Dominant pattern of failure
No recurrence 23 (21.7)
Locoregional 16 (15.1) 25.1 0.044 11.5 0.045
Widespread 36 (34.0) 16.7 4.0
Combined 27 (25.5) 17.5 5.9
Unknown 4 (3.8)
The bold values indicate P values less than 0.05. AWD, alive with disease; NED, no evidence of disease; PDAC, pancreatic ductal adenocarcinoma; SD, standard
deviation; UICC, Union for International Cancer Control.
survival (Table 1 and Fig. 4). Similarly, loss of p16 (P = 0.015) loss of Smad4/Dpc4 immunolabeling was an independent predictor
and Smad4/Dpc4 (P < 0.001) immunolabeling was associated with (hazard ratio 1.888, P = 0.015) (Supplemental Digital Content 2,
a significantly shorter disease-free survival (Table 1 and Supplemen- available at http://links.lww.com/SLA/A348).
tal Digital Content 1, available at http://links.lww.com/SLA/A349).
There were borderline significant differences in abnormal labeling Clinicopathological Characteristics Versus Pattern
of p53 with regard to overall survival (P = 0.060) and disease- of Disease Progression
free survival (P = 0.051). There was no difference of survival be- We determined the relationships between the clinicopatholog-
tween the types of abnormal p53 immunolabeling (virtual absence ical factors (tumor size, lymph node metastasis, margin status, dif-
of p53 immunolabeling vs. robust nuclear accumulation of p53 im- ferentiation, lymphatic invasion, and vascular invasion), and the first
munolabeling: overall survival, P = 0.528; disease-free survival, site of recurrence and the dominant pattern of disease progression.
P = 0.643). Among them, only lymphatic invasion was significantly associated
Next, based on the number of altered genes, we classified with locoregional recurrence as the primary sign of recurrence (P
the patients into 4 groups: zero gene (n = 4), 1 gene (n = 18), 2 = 0.011). No clinicopathological factors significantly impacted upon
genes (n = 49), and 3 genes (n = 35). Statistical analysis compar- the pattern of recurrence. The disease-free survival duration in 38
ing the zero gene and other groups was not performed because of patients who presented with distant metastases as the first sign of
the small number of patients. Kaplan-Meier survival analysis showed recurrence was very short (median 4.0 months) and this rapid pro-
that there was a significant difference between 1-gene and 3-genes gression strongly suggests that occult metastatic disease was present
group (P < 0.001) and between 2-genes and 3-genes group (P = at the time of surgery.
0.002) in overall survival (Fig. 5). The patients with 2 altered genes
had shorter overall survival than those with 1 altered gene but without Immunohistochemical Labeling of p16, p53, and
statistical significance (P = 0.076). Similarly, Kaplan-Meier survival
analysis for disease-free survival showed that there was a signif-
Smad4/Dpc4 Versus Pattern of Disease Progression
icant difference between 1-gene and 2-genes group (P = 0.015), We also compared the immunohistochemical findings of p16,
between 1-gene and 3-genes group (P < 0.001) and between 2-genes p53, and Smad4/Dpc4 with the first sign of recurrence (Table 2) and
and 3-genes group (P = 0.007) (Fig. 5). The increasing number found that loss of p16 immunolabeling was significantly associated
of the altered genes robustly reflected major differences in survival with distant metastases (locoregional recurrence vs. distant metas-
outcome. tases, P = 0.005). Similarly, comparing the immunohistochemical
findings for p16, p53, and Smad4/Dpc4 with regard to the domi-
nant pattern of disease progression, loss of p16 immunolabeling was
Relationship Between Prognostic Factors and significantly associated with widespread metastases (locoregional re-
Survival by Multivariate Analysis currence vs. widespread metastases, P < 0.001).
Multivariate models using Cox proportional hazards analysis
were conducted with the parameters that were significant at the P < DISCUSSION
0.1 level on univariate analysis using log-rank tests. Multivariate anal- It is accepted that various pathological factors, including tumor
ysis demonstrated loss of Smad4/Dpc4 immunolabeling (hazard ratio differentiation, tumor size, lymph node status, lymphatic/vascular in-
2.045, P = 0.014) to be an independent prognostic factor for overall vasion, and resection margin involvement, influence outcome after
survival (Table 3). There were borderline significant relationships of PDAC resection.33 However, “preoperative” identification of patients
overall survival with the T factor (hazard ratio 2.167, P = 0.056) with a poor prognosis is desirable to aid appropriate clinical decision-
and abnormal p53 immunolabeling (hazard ratio 1.978, P = 0.051). making. In the present study, univariate analyses revealed that tradi-
Multivariate analysis for disease-free survival duration showed that tional clinicopathological factors, including T factor (T3 vs. T1/T2),
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Oshima et al Annals of Surgery r Volume 258, Number 2, August 2013
TABLE 2. Clinicopathological Parameters and Immunohistochemical labeling of p53, p16 and Smad4/Dpc4 (n = 106)
p16 Immunohistochemistry p53 Immunohistochemistry Smad4/Dpc4 Immunohistochemistry
Positive Negative Abnormal Positive Negative
Variable (Intact) (%) (Loss) (%) P Value Normal (%) (%) P Value (Intact) (%) (Loss) (%) P Value
Tumor size
Mean (mm) 32.1 30.4 0.539 28.1 31.6 0.300 26.5 33.9 0.006
≤ 20 mm 6 (17.1) 18 (25.4) 0.342 7 (35.0) 17 (19.8) 0.143 13 (31.0) 11 (17.2) 0.098
> 20 mm 29 (82.9) 53 (74.6) 13 (65.0) 69 (80.2) 29 (79.0) 53 (82.8)
Lymph nodes
Negative 15 (42.9) 19 (26.8) 0.095 8 (40.0) 26 (30.2) 0.393 20 (47.6) 14 (21.9) 0.006
Positive 20 (57.1) 52 (73.2) 12 (60.0) 60 (69.8) 22 (52.4) 50 (78.1)
Margin status
R0 29 (82.9) 46 (64.8) 0.055 17 (85.0) 58 (67.4) 0.120 31 (73.6) 44 (68.8) 0.575
R1 6 (17.1) 25 (35.2) 3 (15.0) 28 (32.6) 11 (26.2) 20 (31.2)
Differentiation
Well 22 (62.9) 39 (54.9) 0.669 17 (85.0) 44 (51.2) 0.022 26 (61.9) 35 (54.7) 0.695
Moderate 8 (22.9) 22 (31.0) 2 (10.0) 28 (32.6) 10 (23.8) 20 (31.2)
Poor 5 (14.3) 10 (14.1) 1 (5.0) 14 (16.3) 6 (14.3) 9 (14.1)
Lymphatic invasion
Negative 13 (37.1) 11 (15.5) 0.012 6 (30.0) 18 (20.9) 0.383 14 (33.3) 10 (15.6) 0.033
Positive 22 (62.9) 14 (70.0) 68 (79.1) 60 (84.5) 28 (66.7) 54 (84.4)
Vascular invasion
Negative 7 (20.0) 7 (9.9) 0.147 5 (25.0) 9 (10.5) 0.084 7 (16.7) 7 (10.9) 0.394
Positive 28 (80.0) 64 (90.1) 15 (75.0) 77 (89.5) 35 (83.3) 57 (89.1)
T-factor (UICC)
T1 3 (8.6) 4 (5.6) 0.648 3 (15.0) 4 (4.7) 0.244 5 (11.9) 2 (3.1) 0.195
T2 5 (14.3) 7 (9.9) 2 (10.0) 10 (11.6) 5 (11.9) 7 (10.9)
T3 27 (77.1) 60 (84.5) 15 (75.0) 72 (83.7) 32 (76.2) 55 (85.9)
T4 0 0 0 0 0 0
Stage (UICC)
IA 3 (8.6) 1 (1.4) 0.107 2 (10.0) 2 (2.3) 0.226 4 (9.5) 0 0.018
IB 3 (8.6) 2 (2.8) 2 (10.0) 3 (3.5) 3 (7.1) 2 (3.1)
IIA 9 (25.7) 15 (21.1) 4 (20.0) 20 (23.3) 12 (28.6) 12 (18.8)
IIB 20 (57.1) 53 (74.6) 12 (60.0) 61 (70.9) 23 (54.8) 50 (78.1)
Locoregional recurrence
Present 18 (52.9) 38 (56.7) 0.718 6 (31.6) 50 (61.0) 0.020 20 (48.8) 36 (60.0) 0.265
Absent 16 (47.1) 29 (43.3) 13 (68.4) 32 (39.0) 21 (51.2) 24 (40.0)
Unknown 1 4 1 4 1 4
First sign of recurrence
No recurrence 12 11 7 16 16 7
Locoregional 13 (56.5) 14 (23.7) 0.015 5 (38.5) 22 (31.9) 0.450 8 (30.8) 19 (33.9) 0.640
Distant metastases 6 (26.1) 32 (54.2) 7 (53.8) 31 (44.9) 11 (42.3) 27 (48.2)
Synchronous 4 (17.4) 13 (22.0) 1 (7.7) 16 (23.2) 7 (26.9) 10 (17.9)
Unknown 0 1 1 1
Dominant pattern of failure
No recurrence 12 11 7 16 16 7
Locoregional 10 (45.5) 6 (10.5) 0.001 2 (15.4) 14 (21.2) 0.791 4 (16.0) 12 (22.2) 0.074
Widespread 5 (22.7) 31 (54.4) 7 (53.8) 29 (43.9) 8 (32.0) 28 (51.9)
Combined 7 (25.9) 20 (35.1) 4 (30.8) 23 (34.8) 13 (52.0) 14 (25.9)
Unknown 1 3 0 4 1 3
The bold values indicate P values less than 0.05. UICC, Union for International Cancer Control.
lymphatic invasion, and lymph node metastasis, predict outcome as encodes a critical transcription factor involved in the TGF-β signal
expected, and furthermore that p16 and Smad4/Dpc4 immunolabeling pathway, whose dysregulation promotes the epithelial mesenchy-
correlated significantly with the prognosis. Multivariate analyses in- mal transition, an event that occurs normally during embryonic
dicated that loss of Smad4/Dpc4 immunolabeling was an independent development and is thought to be a critical contributor to tumor
predictor of shorter overall and disease-free survival. In addition, loss invasiveness and metastasis.39–41 Furthermore, it has been recently
of p16 immunolabeling was significantly associated with widespread documented that mutations of SMAD4/DPC4 predict survival15 and
metastases. loss of Smad4/Dpc4 immunolabeling correlates with patterns of
The clinical implications of the genetic changes linked tumor progression.30,42 We here clarified clinical significance of
to prognosis may still be conflicting and controversial.15,16,34–36 Smad4/Dpc4 immunolabeling loss as a predictive marker of lymph
SMAD4/DPC4 was here found to be inactivated in 64 (60.4%) of node metastasis and postoperative survival outcome in a population
the 106 PDACs, similar to the value (55%) reported by Hahn et al of patients independent from those initially described,15,30 although
(homozygous deletion in 35% of cases and loss of 1 allele coupled it should be stressed that it was not associated with the pattern of
with an intragenic mutation in the second allele in 20%).37,38 It tumor progression.
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Annals of Surgery r Volume 258, Number 2, August 2013 p16, p53 and Smad4 in Pancreatic Cancer
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Oshima et al Annals of Surgery r Volume 258, Number 2, August 2013
FIGURE 5. Kaplan-Meier survival curves for patients after surgery for pancreatic ductal adenocarcinoma demonstrating relation-
ships of the number of altered genes with postoperative overall survival (A) and disease-free survival (B).
Prospective validation of the expression of p16, p53, Smad4/Dpc4 (14.1%) died as a result of complications of locoregional tumor pro-
and their combinations as prognostic biomarkers in a large series of gression, a lower proportion than in the rapid autopsy series from
patients is clearly warranted. The present study also focused on the the Johns Hopkins Medical Institutions,30 probably because of differ-
first sign of recurrence and patterns of failure after surgery. Among ences in study populations. In the present study, however, one-third of
the 63 patients who died of PDAC with available information, 9 patients were classified into the combined pattern (both locoregional
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Annals of Surgery r Volume 258, Number 2, August 2013 p16, p53 and Smad4 in Pancreatic Cancer
recurrence and widespread metastases), which indicates that it might 9. Yachida S, Jones S, Bozic I, et al. Distant metastasis occurs late during the
be practically difficult to classify patterns of progression only with genetic evolution of pancreatic cancer. Nature. 2010;467:1114–1117.
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