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Molecular Oncology, Markers, Clinical Correlates |
Department of Pathology, The Gade Institute [H. B. S., L. A. A.] and Department of Gynecology and Obstetrics [H. B. S.], Haukeland University Hospital, N-5021 Bergen, Norway, and Department of Human Genetics, The University of Chicago, Chicago, Illinois 60637 [H. B. S., S. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In endometrial carcinomas, p16 alterations seem to be rare, although a few studies have been published. None of these studies, however, has focused on prognostic relevance of p16 alterations. Wong et al. (13) found homozygous deletions in only 1 of 41 tumors (2%). In the recent report by Nakashima et al. (14) , homozygous deletions were found in 1 of 38 tumors (3%), and p16 gene mutations were found in 2 of 38 tumors (5%). Peiffer et al. (15) found p16 gene mutations in 2 of 34 tumors (6%), whereas Hatta et al. (16) did not find any alterations in 15 endometrial carcinomas studied. In the recent report by Milde-Langosch et al. (17) , one point mutation and no deletions were found in 36 tumors (3%), despite negative or minimal nuclear staining for p16 protein in 74% of the tumors. Another previous immunohistochemical study of p16 protein expression in endometrial carcinomas showed that 66% of the cases were negative for nuclear staining (18) , whereas Nakashima et al. (14) found negative nuclear staining in 19% of the tumors in their immunohistochemical study. The recent study of Nakashima et al. (14) reported no cases with p16 promoter methylation in the 26 cases studied. With this background, the aim of our study was to assess the pattern and prognostic impact of p16 protein expression and p16 promoter methylation in a population-based, large series of endometrial carcinomas and to correlate the findings with data on clinicopathological variables and other tumor markers.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the potential selection bias in the patient population
with frozen tumor tissue available, patient age, FIGO stage,
histological type and grade, treatment, and survival were compared for
these patients (n = 138) with those of the rest of the
patients from Hordaland County treated for endometrial carcinoma during
the same period from whom fresh tumor tissue was not available
(n = 161). Curative surgical treatment was more often
possible in the group with fresh tumor tissue available (92%) than in
the rest of the population (77%; P < 0.001,
2 test). This is in accordance with the
current practice in the area; women ineligible for curative treatment
due to either advanced age or serious intercurrent or extensive disease
are less often referred to the University Hospital, where fresh tissue
was collected during primary surgery. With this exception, there were
no significant differences in the other patient characteristics. Among
the patients treated for cure, no significant difference in survival
was found for the patients with fresh tumor tissue available compared
with the rest of the patients.
Immunohistochemistry.
Immunohistochemical staining was performed on formalin-fixed,
paraffin-embedded specimens. H&E-stained sections were used to select
the most cellular part of the tumor and the area with lowest
architectural differentiation in the case of heterogeneity. Five-µm
sections from selected areas were used for immunohistochemistry, and
the staining procedures and results for microvessel density counts and
Ki-67, p53, and p21 expression have been described previously
(20
, 22 , 23)
.
p16 Expression.
For estimation of p16 protein expression, the sections were incubated
overnight at room temperature with polyclonal antibody SC-468 (Santa
Cruz Biotechnology, Santa Cruz, CA) diluted 1:250 in PBS. In every run,
positive control sections known to express p16 were included. In
addition, nuclei in stromal cells were used as internal positive
controls. Negative controls obtained by omitting the primary antibody
were also included in every run. Nuclear p16 staining was recorded by a
semiquantitative and subjective grading, considering both the intensity
of staining and the proportion of tumor cells in the selected section
showing an unequivocal positive reaction as described previously for
p53 (20
, 23)
. Intensity was recorded as 0 (no staining) to
3 (strong staining) whereas the percentage of nuclear staining was
recorded as 0 (no tumor cells positive), 1 (positive staining in <10%
of the tumor cells), 2 (positive staining in 10–50% of the tumor
cells), and 3 (positive staining in >50% of the tumor cells). A
staining index was calculated as the product of staining intensity and
staining area. The tumors were categorized as moderate/high expressing
(staining index 
4) or absent/minimal expressing (staining
index < 4) tumors. An equivalent staining index was also
estimated for the cytoplasmic staining by scoring the cytoplasmic
staining intensity and area. On the basis of previous studies
(24)
, we especially wanted to see whether clearly reduced
nuclear p16 expression was of prognostic importance. We therefore
compared the group with absent or minimal nuclear staining (staining
index < 4) with the rest (staining index 4). However,
additional survival analyses categorizing the nuclear staining into
smaller groups according to each level of the index showed a similar
survival for the subgroups with staining indices < 4, which
differed from that of the subgroups with staining indices 4,
further justifying this cutoff value.
To investigate the observer reproducibility of the p16 staining
reading, a subset of the sections was investigated two times by the
same observer (H. B. S.) and by two different observers (H. B. S.
and L. A. A.). The exact intraobserver agreement for nuclear staining
index 4 versus nuclear staining index < 4 was
96% (
= 0.89). The corresponding exact interobserver agreement
was 93% ( = 0.83). In addition to using internal and external
positive controls as described above, the immunohistochemical staining
method was further validated by repeating the staining procedure for 25
sections. This gave an 88% exact agreement for the nuclear staining
index (4 versus <4, = 0.75).
In a subset of this population-based series, DNA ploidy, S-phase fraction, steroid receptor concentration, and p16 methylation status were assessed on frozen tissue specimens taken during the primary operation from a site considered macroscopically to be representative for the tumor. These samples were divided into three parts: (a) one for receptor analyses (estradiol/progesterone receptors); (b) one for flow cytometric and molecular studies; and (c) one for routine histological examination to confirm that the tissue contained at least 50% tumor cells. Technical details regarding measurements of DNA ploidy, S-phase fraction, and steroid receptor concentration have been presented previously (22 , 25) .
DNA Isolation.
DNA was isolated from the 138 frozen tumors by digestion with
proteinase K in STE and 10% SDS, followed by a standard
phenol-chloroform extraction and ethanol precipitation.
Methylation Analysis.
The methylation status of the p16 gene in the tumors was
determined by M-PCR as described by Herman et al.
(26)
. Briefly, genomic tumor DNA was treated with sodium
bisulfite, which converts all unmethylated cytosine residues to uracil,
which is then converted to thymidine in the subsequent PCR. Two primer
sets were used to amplify the region of interest: (a) one
specific for the unmethylated sequence where CpG sites are modified by
bisulfite treatment (p16-U1); and (b) one specific for the
methylated sequence where CpG sites remain unmodified by bisulfite
treatment (p16-M1; Table 1
). We used the
same primer sequences described by Herman et al.
(26)
for p16-M1 and the p16-U1 reverse primer, which
localize to regions in and around the transcription start site of the
p16 gene, a region previously reported to be correlated with
loss of gene expression (7
, 26)
. All of the tumors were
investigated with the p16-M1 primer set at least twice.
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. The M-PCR was repeated with these two primer
sets for all 26 cases with fresh tumor tissue available showing absent
or minimal nuclear p16 protein expression and for an equal number of
tumors showing nuclear p16 protein expression.
The PCRs were carried out in a 25-µl volume containing 1x PCR
buffer II (Perkin-Elmer, Foster City, CA), 2.5 mM
MgCl2, 200 µM deoxynucleotide
triphosphate, 0.5 µM of each PCR primer, 0.5 unit of
AmpliTaq Gold (Perkin-Elmer), and approximately 40 ng of
bisufite-modified DNA. Amplification was carried out in a Perkin-Elmer
model 9600 thermocycler at 95°C for 10 min and cycled at 94°C for
45 s, 30 s at the annealing temperature listed in Table 1
,
and 30 s at 72°C (40 cycles for primer sets U1, M1, and M2 and
35 cycles for primer set M3), followed by a 10-min extension at 72°C.
The positive control (C2) for the methylated primer sets consisted of
lymphocyte DNA treated in vitro with excess SssI
methyltransferase (New England Biolabs, Beverly, MA), which generated
DNA that was completely methylated at all CpG sites. The positive
control (C1) for the unmethylated primer set consisted of lymphocyte
DNA from normal individuals; however, because the tumors also contain
normal cells, amplification of the unmethylated sequence is
expected to be present for all tumors. Reactions containing untreated
DNA (C3) and no DNA (C4) were included as negative controls for all
primer sets. The amplification products were separated on a 3% agarose
gel and visualized by ethidium bromide staining and UV illumination.
Follow-Up.
The median follow-up period for the survivors was 9 years (range, 4–15
years). None of the patients was lost due to insufficient follow-up
data. Information about survival was obtained from medical records and
correspondence with the primary physician. The data were cross-checked
with information from the Cancer Registry of Norway, which is matched
against the Register of Deaths at Statistics Norway.
Statistics.
Comparisons of groups were performed using the
2 test. Reproducibility was assessed using
statistics. Univariate survival analyses of time to death due to
endometrial carcinoma (cause-specific death) were performed using the
product-limit procedure (Kaplan-Meier method), with the time of the
primary operation as the entry date. Patients who died of other causes
were censored at the date of death. The Mantel-Cox test was used to
compare the survival curves for groups of patients defined by
categories of each variable. The variables with significant impact on
survival in univariate analyses (P 
0.10) were
further examined by log-minus-log plot to decide how these variables
should be incorporated in the multivariate Cox PH regression model.
Hazard ratios were estimated as a measure of effect. Tests for
interactions were carried out for the variables with independent impact
on survival in the multivariate analysis. Data were analyzed using the
SPSS software package (SPSS Inc., Chicago, IL).
The research has been approved by the Norwegian Data Inspectorate and the Institutional Review Board at the University of Chicago (Protocol 9457).
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4).
Absent or minimal nuclear p16 protein expression was associated with
increased patient age at primary operation, high FIGO stage, serous
papillary or clear cell histological types, high histological grade,
aneuploidy, low estradiol and progesterone receptor concentrations,
high expression of Ki-67, high microvessel density, and strong nuclear
p53 protein expression, as shown in Table 2
. The mean Ki-67 expression was 40%
positive nuclei, and the MVDmean was 97
microvessels/mm2 among the tumors with
absent/minimal nuclear expression of p16 protein (staining index < 4), compared with 23% and 71 microvessels/mm2
among the tumors with moderate/high nuclear expression of p16 protein
(index 4).
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Nuclear p16 expression was studied further in Cox PH regression
analysis, together with age at primary operation, FIGO stage,
histological type, histological grade, DNA ploidy, S-phase fraction,
progesterone receptor concentration, microvessel density, Ki-67, p53,
and p21 expression (all with P 0.10 in univariate
analysis). As shown in Table 3
, nuclear
p16 expression had an independent prognostic impact in addition to FIGO
stage, patient age, Ki-67 expression, and microvessel density. The
other variables lost their prognostic impact when nuclear p16
expression was added to the Cox model.
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Of the 286 cases studied for p16 protein expression, fresh tumor tissue
was available and used for M-PCR in 138 cases. This group of patients
included 26 patients (19%) with absent or minimal nuclear expression
of p16 protein (staining index < 4), whereas 112 patients (81%)
had moderate or high p16 expression (index 4). Only one tumor
(0.7%) was found to be methylated in the p16 promoter
region (Fig. 2)
. This result was
confirmed with three different primer sets. This tumor lacked nuclear
p16 protein expression as well.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because hypermethylation of p16 has been documented in a number of other tumor types and appears to be a common mode of inactivation of this gene (6 , 7) , we wanted to determine the role of p16 promoter hypermethylation in this group of endometrial carcinoma patients, particularly in the group with loss of protein expression. One recent but smaller report found no hypermethylation of the p16 promoter region in 26 endometrial carcinomas studied with a methylation-sensitive restriction enzyme method (14) . Our finding that only 1 of the 138 cases studied showed hypermethylation for the p16 promoter region (0.7%) supports that this is not the predominant mechanism of p16 inactivation in these tumors. It is possible that deletions and/or mutations of the p16 gene may play a more significant role in those tumors with minimal or low protein expression, but the incidence of deletions and mutations in our group of endometrial carcinoma tumors remains to be determined. However, previous studies of p16 alterations in endometrial carcinoma appear to indicate that deletions and mutations of this gene are rare (13 , 15, 16, 17) . Therefore, to date, the mechanism of p16 inactivation, as indicated by a lack of p16 protein expression, remains largely unknown for endometrial carcinomas.
Loss of p16 protein expression was significantly related to a subgroup of aggressive endometrial carcinomas and poor prognosis. Thus, absent or minimal nuclear staining was associated with increased age at treatment, higher FIGO stage, serous papillary or clear cell histological types, high histological grade, and aneuploid tumors. Furthermore, there were strong correlations with increased tumor cell proliferation, as measured by the expression of Ki-67, supporting the role of intact p16 protein as a cell cycle inhibitor (27) . Interestingly, lack of nuclear p16 expression was also significantly associated with increased intratumor angiogenesis, as measured by factor VIII-related microvessel density. However, the reason for this is not clear. Finally, there was a statistically significant association between altered nuclear p16 and p53 expression. In 13 patients (7%), alterations were indicated in both of these major tumor suppressor pathways. This has also been indicated in studies of other tumors (28 , 29) . In 27% of the cases, alterations were present in either the p16 or the p53 systems, whereas 73% of the tumors showed no apparent alterations, as judged by immunohistochemical expression of the protein products.
In endometrial carcinoma, the prognostic impact of traditional clinicopathological variables is used to determine whether hysterectomy alone is likely to be curative (30 , 31) . In addition, a number of studies have identified aneuploidy and high S-phase fraction as negative prognostic factors (25 , 32) . Although markers for angiogenesis, proliferative activity, and selected cell cycle regulators have recently been found to add prognostic information (20 , 22 , 23 , 32 , 33) , there is still a need for more specific and even better prognostic markers to avoid overtreatment of low-risk groups and to ensure that patients with highly aggressive tumors receive adequate postoperative treatment (30 , 34) . The prognostic impact of alterations of the p16 system, which represents a major tumor suppressor pathway, has not previously been addressed in endometrial carcinomas. Survival analysis showed that lack of nuclear p16 protein expression was significantly associated with a markedly increased risk of disease-related death. Even in multivariate analysis including the traditional clinicopathological variables as well as markers for cell cycle regulation, proliferation, and angiogenesis, nuclear p16 protein expression had a strong and independent prognostic impact. When nuclear p16 expression was included in the Cox model, age, FIGO stage, Ki-67 expression, and intratumor microvessel density still had an independent prognostic impact, whereas p53 expression, for example, lost its importance.
In conclusion, we have shown that lack of nuclear p16 protein expression in endometrial carcinoma, as an indication of p16 inactivation, is associated with a subset of aggressive tumors and has an independent prognostic impact. The mechanism of p16 inactivation is not clear, but promoter region methylation seems to be very rare (only 0.7% in our present series). Because the immunohistochemical data indicate a higher frequency of inactivation, other genetic alterations or nongenetic mechanisms for inactivation should be further studied.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by The Norwegian Cancer Society Grant
D96032/D94070 and NIH Grant CA81652-01. 
2 To whom requests for reprints should be
addressed, at Department of Gynecology and Obstetrics, Haukeland
University Hospital, N-5021 Bergen, Norway. Phone: 47-55-97-42-00; Fax:
47-55-97-49-68; E-mail: hesa{at}haukeland.no
3 The abbreviations used are: FIGO, International
Federation of Gynecology and Obstetrics; M-PCR, methylation-specific
PCR; MVDmean, mean intratumoral microvessel density; PH,
proportional hazards.
Received 5/18/99; revised 10/ 1/99; accepted 10/ 7/99.
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REFERENCES |
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