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Molecular Oncology, Markers, Clinical Correlates |
Department of Pathology, The Gade Institute, Haukeland University Hospital, N-5021 Bergen, Norway
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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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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Complete information on patient survival and time and cause of death was available in all 202 cases, and information on recurrence-free survival was available in 167 patients.
Clinicopathological Variables
The following variables were recorded: (a) date of
histological diagnosis; (b) sex; (c) age at
diagnosis; (d) anatomical site of the primary tumor; and
(e) presence of local (skin), regional (lymph node), or
distant metastases at diagnosis. The H&E-stained slides were
reexamined, and the following histological features were also included:
(a) tumor thickness according to Breslow (27)
;
(b) histological tumor diameter (measured horizontally on a
central cross-section); (c) level of invasion according to
Clark et al. (28)
; (d) microscopic
ulceration; and (e) vascular invasion (each case was
examined closely using both H&E- and factor VIII-stained slides).
Staging was done according to the
AJCC3
pTNM
(tumor-node-metastasis) staging system, including both clinical and
histological data.
Immunohistochemistry
p16.
The immunohistochemical staining was performed on formalin-fixed and
paraffin-embedded archival tissue. Thin sections (5 µm) were
incubated with the polyclonal p16 antibody SC-468 (Santa Cruz
Biotechnology, Santa Cruz, CA) overnight at room temperature. For the
cases from 1981–1989, we used the slides that had been stained
previously with an antibody dilution of 1:500 (19)
. Using
the same protocol for cases from 1990–1997, an antibody dilution of
1:200 was determined to obtain an equivalent staining reaction by
comparing positive control slides from identical specimens. This
difference is probably due to the fact that a different antibody batch
was used for the last part of the series, although minor differences in
other technical details like routine fixation procedures cannot be
excluded. It has been suggested by others that antigen retrieval by
microwave pretreatment might suppress p16 immunoreactivity
(29)
, and protocols with or without retrieval were tested.
Strong staining (in positive controls) was observed without retrieval
pretreatment, whereas clearly weaker staining was present after
microwave treatment, which was therefore not used in our study. Nuclear
staining in stromal cells and adnexal glands was used as a positive
internal control, whereas cases of endometrial carcinoma, known
to be positive for p16 protein expression, were used as positive
external controls.
p53.
The p53 staining protocol included microwave antigen retrieval (10 min
at 750 W and 3 x 5 min at 500 W) and incubation for 1 h at
room temperature with the DO-7 monoclonal antibody (M-700; DAKO,
Copenhagen, Denmark) diluted 1:100 (30)
.
Ki-67.
After microwave antigen retrieval (10 min at 750 W and 4 x 5 min
at 500 W), the sections were incubated for 1 h at room temperature
with the polyclonal Ki-67 antibody (A-047; DAKO) diluted 1:50
(30)
.
The staining procedures were performed on DAKO TechMate 500 slide processing equipment, using the standard avidin-biotin complex method. Finally, the peroxidase was localized by the 3-amino-9-ethylcarbazole (AEC) peroxidase reaction, using Harris hematoxylin as a brief (30 s) counterstain. Negative controls were obtained by omitting the primary antibody.
Evaluation
Immunohistochemical staining of p16 and 53 proteins was recorded
as described previously (19)
, considering both the
staining intensity and the proportion of positive tumor cells.
Intensity was graded as follows: (a) 0, no staining;
(b) 1, weak staining; (c) 2, moderate staining;
and (d) 3, strong staining. Area was graded as follows:
(a) 0, no positive tumor cells; (b) 1, <10%
positive tumor cells; (c) 2, 10–50% positive tumor cells;
and (d) 3, >50% positive tumor cells. Staining index was
computed as follows: staining index = staining intensity x
positive area.
Combined examination of p16 expression in both nuclear and cytoplasmic compartments has been used by some researchers (14) . Because cytoplasmic staining is controversial and regarded as nonspecific by others (15 , 18 , 29) , we decided to examine cytoplasmic and nuclear staining separately.
Ki-67 staining was assessed according to the approach of Weidner et al. (31) . Briefly, the tumors were scanned at low magnification (x40 and x100) to identify the areas of most intense nuclear staining (hot spots). The percentage of immunoreactive tumor cell nuclei (the proliferative rate) was then calculated by counting at least 500 cells at x1000 magnification within the selected areas.
Follow-Up
Hospital records were used to obtain follow-up information,
including the presence and type of recurrences as well as the time
until recurrence. In some cases, general practitioners responsible for
the follow-up of the patient were contacted. Information about the
cause and date of death was obtained from the Cancer Registry of Norway
and Statistics Norway. Complete information on patient survival and
time and cause of death was available in all 202 cases. Last date of
follow-up was December 18, 1998, and median follow-up time for all
survivors (total survival) was 76 months (range, 13–210 months).
Clinical follow-up (with respect to recurrences) was not carried out in
14 patients (predominantly older patients), and 21 patients were not
treated with complete local excision. Thus, recurrence-free survival
was available in 167 patients.
During the follow-up period, 69 patients (34%) died of malignant melanoma, and 39 (19%) died of other causes. Of the 167 radically treated patients with data on recurrence-free survival, 74 (44%) had recurrent disease.
Statistics
Analyses were performed using the SPSS statistical package
(32)
. Associations between different categorical variables
were assessed by Pearson’s 
2 test. Continuous
variables not following the normal distribution were compared between
two or more groups using the Mann-Whitney U or
Kruskal-Wallis H tests. Wilcoxon’s signed ranks test and
paired samples t test were used to compare related samples.
The relationship between tumor thickness and Ki-67 expression was
further assessed using linear regression analysis. Univariate analyses
of time to death due to malignant melanoma or time to recurrence
(recurrence-free survival) were performed using the product-limit
procedure (Kaplan-Meier method), with date of histological diagnosis as
the starting point. Patients who died of other causes were censored at
the date of death. Differences between categories were tested by the
log-rank test. The influence of covariates on patient survival and
recurrence-free survival was analyzed by the proportional hazards
method (33)
, including all variables with a
P 
0.15 in univariate analyses, and tested by the
likelihood ratio test. In the multivariate analysis, categories that
had a similar prognosis in univariate analysis or were too small to be
analyzed separately were merged. Model assumptions were tested by
log-minus-log plots. Estimated HR, 95% confidence interval for HR, and
Ps are given in the tables.
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RESULTS |
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was most often homogeneous throughout
the tumor. Some cases showed a more heterogeneous staining pattern
(mosaic type), with groups of positive and negative tumor cells side by
side. Cytoplasmic staining was often observed in cases lacking nuclear
staining, and the indices for cytoplasmic and nuclear staining are
shown separately in Table 1
. There was
considerable variation in staining intensity for both compartments, and
the most intensely stained sections most often showed combined
cytoplasmic and nuclear staining in both primary tumors and metastases.
Of all primary tumors, 45% showed no nuclear staining or minimal
nuclear staining for p16 protein (staining index, 0–1), as compared
with 77% of the metastases (Wilcoxon’s signed ranks test,
P = 0.001; Table 1
). A similar difference between
primary tumors and corresponding metastases was observed for
cytoplasmic staining (Table 1)
.
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; Fig. 2
),
and recurrence-free survival was also significantly decreased in this
subgroup of patients (P = 0.007). No significant
association was present between cytoplasmic p16 expression and patient
outcome.
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; Table 1
). A
high p53 staining (index 
6) was found in 11% of the
primary tumors and was significantly more frequent in the head/neck
area when compared with other sites (20% versus 8%;
P = 0.04). There was no significant difference in p53
expression between primary tumors and corresponding metastases. No
significant association was found between p53 and other
clinicopathological variables as well as p16 expression. There was a
tendency toward lower Ki-67 expression (median, 23%) in cases with an
absence of nuclear p53 expression compared with all p53-positive cases
(median, 28%), although this trend was not statistically significant
(Mann-Whitney U test, P = 0.12).
Furthermore, there was no significant difference in Ki-67 staining
between cases with strong versus weak/negative p53
expression.
In this extended series, we found a significantly improved survival for
those cases (16%) without any p53 protein expression in the melanoma
cells (Table 2
; Fig. 2
; P = 0.0057) when compared with
the rest of the cases, i.e., cases with weak, moderate, or
strong p53 expression. In contrast, strong p53 expression (staining
index 6) had no significant prognostic impact when compared
with the remaining cases (staining index, 0–4; P =
0.4). The presence of p53 expression (positive versus
negative) did not significantly predict reduced recurrence-free
survival (P = 0.12), with 10-year recurrence-free
survival rates of 35% and 60% for cases with and without p53
expression, respectively.
Ki-67 Protein Expression.
The staining pattern for Ki-67 varied in different lesions. Some of the
tumors exhibited focal aggregates of immunoreactive nuclei, whereas
other tumors showed diffuse, more homogeneous distribution of positive
nuclei. However, it was possible to identify hot spots of stained
nuclei in all cases with material available, and these hot spots were
most often found in the periphery of the tumor, i.e., near
the invasive front (tumor basis) or near the epidermis.
The median percentage of Ki-67-positive nuclei within the selected hot
spot areas was 27% (range, 1–79%). The proportion of positively
stained nuclei was significantly higher in the metastases than in the
corresponding primary tumors, with a median count of 43% (paired
t test, P < 0.0001). Increased Ki-67
expression was significantly correlated with increased tumor thickness
(linear regression analysis, r = 0.32;
P < 0.0001; Fig. 3
),
increased histological tumor diameter (linear regression analysis,
r = 0.28; P < 0.0001), and increased
Clark’s level of invasion (levels 2–4 versus level 5;
t test, P = 0.02). Lesions located on the
trunk and on the head and neck exhibited significantly higher tumor
cell proliferation (mean, 32%) than lesions located on the extremities
(mean, 24% Ki-67-positive nuclei; t test, P = 0.004). Furthermore, tumor ulceration (t test,
P < 0.0001) and the presence of vascular invasion
(t test, P = 0.02) were also significantly
related to higher Ki-67 expression.
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;
P < 0.0001) and recurrence-free survival
(P = 0.0001) were observed in patients with the highest
values. In later analyses, the patients were divided into two groups by
the lower quartile, as illustrated in Fig. 2
, because the upper three
categories had a similar prognosis.
Multivariate Analysis of Patient Survival.
The following variables, which represent features of the primary tumor
that were all either significant or of borderline significance in
univariate analysis (P 0.15), were included in the
multivariate analysis: (a) anatomical site;
(b) tumor thickness; (c) Clark’s level of
invasion; (d) tumor ulceration; (e) vascular
invasion; and (e) p16, p53, and Ki-67 expression. As a
well-established prognostic factor, tumor thickness was included as the
standard measure of tumor size. Only cases with complete information on
all variables were included in multivariate analysis (n = 187). Anatomical site, Clark’s level of invasion, vascular invasion,
p16 expression, p53 expression, and Ki-67 expression remained as
independent prognostic factors in the final multivariate model (Table 3)
. Tumor thickness (P =
0.34) was not an independent prognostic variable when Clark’s level of
invasion was included in the model. When Clark’s level of invasion was
not included, both thickness (P = 0.03) and Ki-67
expression (P = 0.001) were significant in the model.
All of these prognostic variables (except thickness) remained
significant when AJCC stage was also included in the model [AJCC
stage: HR (all stages) = 2.2; P = 0.0004] as well
as when analyzed for cases with AJCC stage 2 only
(n = 181).
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).
These variables were found to be significant or of borderline
significance (P 0.15) in univariate analysis.
Anatomical site (HR = 1.9; P = 0.02), tumor
thickness (HR = 2.7; P = 0.0005), Clark’s level
of invasion (HR = 2.6; P = 0.005), vascular
invasion (HR = 3.1; P = 0.0002), p16 expression
(HR = 2.0; P = 0.007), and Ki-67 expression
(HR = 2.9; P = 0.01) had independent prognostic
value in the final model, whereas p53 expression was not significant
(HR = 2.1; P = 0.08). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lack of nuclear p16 staining was significantly associated with the presence of tumor ulceration and vascular invasion, but not with tumor thickness, histological diameter, or Clark’s level of invasion. These findings are in agreement with some previous studies (14 , 17) and at odds with others (15 , 18) . Our data may indicate that p16 reduction might be an early event in a subgroup of melanomas and may not be related directly to tumor size at diagnosis.
In melanoma metastases, the frequency of p16 reduction is reported to be even higher than in primary tumors (14 , 17) . In our series, loss of nuclear p16 expression was found in 77% of the metastases, compared with 45% of the primary tumors. Pairwise analyses showed that p16 staining was significantly decreased in the metastases when compared with the corresponding primary melanomas. This may indicate that p16 reduction might also occur in metastases or, alternatively, that subclones of p16-altered tumor cells are more likely to escape from the primary tumor to survive and proliferate at distant sites. The latter explanation is supported by the significant association between p16 loss and vascular invasion. Our findings are in agreement with the conclusion of Morita et al. (35) reporting loss of p16 protein expression (but not p16 mutation) in analysis of primary melanomas and corresponding metastases.
The present data suggest that CDKN2A (p16INK4a) might be inactivated in a relatively high proportion of cutaneous melanomas, although the mechanisms of this process are not clear. Previous studies have shown a high frequency of 9p21 (the site where the p16 gene is located) deletions (36 , 37) , and genetic alterations are particularly common in melanoma cell lines (1 , 38) . In contrast, studies on primary melanomas initially reported a low frequency of mutations (12 , 13 , 39) , but a recent report by Kumar et al. (9) showed that 26% of microdissected cases had intragenic mutations. One study reported a low frequency (10%) of p16 promoter methylation (40) . Thus, mechanisms other than genetic alterations might be operating.
There was a strong and independent association between absent or minimal p16 protein expression and patient prognosis, indicating a close relationship between p16 status of the primary tumor and its ability to spread. In line with this, a significant association between loss of nuclear p16 expression and vascular invasion was found in our series. These prognostic findings are in agreement with those of other reports (14, 15, 16, 17, 18, 19) , although survival studies are few. Our results are similar to those found for other cancer types, such as pancreatic carcinoma (20 , 21) , squamous cell carcinoma of the esophagus (22) , adult T-cell leukemia (23) , and lung carcinoma (24 , 41) .
Loss of nuclear p16 protein expression was closely correlated with significantly increased tumor cell proliferation as measured by Ki-67 staining, supporting the role of intact p16 protein as an important cell cycle inhibitor. Increased proliferation rate was significantly associated with aggressive tumors, i.e., melanomas located on the trunk, tumors with increased thickness and histological diameter, and the presence of tumor ulceration or vascular invasion. High Ki-67 expression was also significantly and independently associated with decreased survival, in accordance with the findings of a few other studies (42 , 43) . Ki-67 expression was associated to some extent with tumor thickness, but proliferation rate was a stronger prognostic factor and was the only remaining variable of the two in the final multivariate model when Clark’s level of invasion was included.
The frequency and role of various p53 changes in cutaneous melanoma have been controversial, and the relation between mutations and protein overexpression is not clear (44, 45, 46) . In a previous study, we found no consistent association between staining results and mutations in the frequently altered exons 7–8 in 30 cases studied (47) . Furthermore, four of the five primary tumors that were mutated showed only weak/focal staining with the DO-7 antibody, whereas most cases with intermediate/strong immunostaining had no detected mutations. In the present study of vertical growth phase melanomas, 16% of all cases were completely p53 negative in the melanoma cells, whereas 11% showed strong nuclear staining; the other cases had intermediate nuclear reactivity (73%). No significant association was found between p53 and Ki-67 expression, and a major regulatory role of p53 for tumor cell proliferation in these advanced primary melanomas is therefore not likely. Correspondingly, no associations between p53 and measures of tumor size (thickness and diameter) and invasion (Clark’s level) were found. Other studies on cutaneous melanoma have suggested that increased p53 expression might be associated with tumor thickness and tumor cell proliferation (43 , 48 , 49) .
Positivity for p53, either weak or strong, was significantly and independently associated with decreased patient survival in our study when compared with completely negative cases. It is known that positive staining for p53 protein might reflect either gene alterations or aberrant stabilization of the wild-type (nonmutant) protein (50) , but the relative importance of different mechanisms of p53 changes, as well as their individual prognostic significance, is not known for melanomas.
Regarding traditional prognostic factors, multivariate analysis showed an independent importance of anatomical site, Clark’s level of invasion, and vascular invasion, whereas tumor thickness did not enter the final model. If Clark’s level was excluded as a variable, both thickness and tumor cell proliferation by Ki-67 staining were of independent importance. Features such as mitotic frequency and lymphocytic response (51) were not examined in the present study.
In conclusion, our findings indicate that loss of nuclear p16 protein expression identifies an aggressive subset of vertical growth phase melanomas and is associated with significantly increased tumor cell proliferation (Ki-67). Lack of p16 staining independently predicts decreased patient survival. Also, p16 reduction was significantly more frequent in metastases than in the corresponding primary tumors. Finally, p53 positivity, Ki-67 expression, and variables such as anatomical site, Clark’s level of invasion, and vascular invasion were also prognostic markers of independent importance in the final multivariate model.
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FOOTNOTES |
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1 Supported by Norwegian Cancer Society Contract
Grant D94070. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, The Gade Institute, Haukeland
Hospital, N-5021 Bergen, Norway. Phone: 47-55-97-31-82; Fax:
47-55-97-31-58; E-mail: lars.akslen{at}gades.uib.no
3 The abbreviations used are: AJCC, American Joint
Committee on Cancer; HR, hazard ratio.
Received 10/15/99; revised 2/15/00; accepted 2/24/00.
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