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Clinical Investigations |
G. Papanikolaou Research Center of Oncology, Athens 11522, Greece [A. S., M. T., N. C., E. D., C. M. T., T. T.], and 1st Medical Oncology Department and Hormone Receptor Unit, St. Savvas Hospital, Athens 11522, Greece [A. A., J. Y.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In view of the heterogeneity of breast carcinomas, in terms of both their biological profile and their clinical outcome, the need for reliable prognostic parameters is obvious. Classical prognostic factors in primary breast carcinoma are tumor size, nodal status, and distant metastases (TNM status) and age. Histopathology and nuclear grading are also standard prognostic variables (2) . The presence of steroid receptors serves predominantly as an indicator of hormone responsiveness (3) . Ploidy [DNA content (4 , 5) ] and proliferative capacity [S-phase fraction (6) ] are the most well-characterized prognostic factors (7) . These measurements have been shown to predict disease-free survival and OS4 in node-negative and node-positive breast cancer. Additional prognostic indicators such as oncogenes, growth factors, and secretory proteins have been investigated and appear to characterize the behavior of the tumor with respect to differentiation extent, growth rate, and metastatic pattern. The expression of the protease cathepsin D has been reported to be an unfavorable prognostic parameter (8) and has been found to promote invasive growth and metastasis of tumor cells (9) . Overall, there is a need to identify as many cellular parameters as possible that will help define the biological profile of the breast tumor cell.
PAP, whose biological function is the polyadenylation of mRNAs, appears to be regulated within the cell cycle and has been shown to vary greatly in terms of enzymatic activity among normal and neoplastic tissues (10 , 11) . The poly(A) tail found in almost all eukaryotic mRNA is important in enhancing translation initiation and determining mRNA stability (12) . Control of the poly(A) tail synthesis has the potential to be a key regulatory step in gene expression. PAP has been reported to reflect the proliferative activity of the cell, and it is also associated with less differentiated cells (11 , 12) . Most studies report that rapidly proliferating and actively metabolizing lymphocytes have higher levels of PAP activity (13 , 14) . Furthermore, it has been suggested to be a unfavorable prognostic factor in chronic leukemias (15, 16, 17) .
PAP activity has been studied initially in the cytosol of breast tumors from 62 untreated patients, and significant association was observed between high PAP activity values and the TNM stage of the disease, node invasiveness, and c-erbB-2 overexpression (18) .
The objective of the present study was to assess the significance of PAP activity in breast cancer prognosis. We measured PAP activity in the tumor cytosols of 228 breast cancer patients and analyzed the relation of PAP to other clinicopathological variables for the RFS and OS using univariate and multivariate analyses.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The age of patients ranged from 24–96 years; the median age was 62 years. All patients had a histologically confirmed diagnosis of primary breast cancer and received no treatment before surgery. Modified mastectomy (98 patients) and breast-conserving lumpectomy (121 patients) with axillary lymph node dissection were performed on 96% of the patients. For patients who had axillary node dissection, the positivity rate for cancer involvement of lymph nodes was 61.8%. The sizes of the tumors resected during surgery ranged from 0.6–7.2 cm, and the mean and median sizes were 2.9 and 2.8 cm, respectively. Clinical staging was performed according to the Postsurgical International Union against Cancer TNM classification system (19) . Histological grade of the tumors was determined according to criteria reported by Bloom and Richardson (20) and was known for 224 patients. Postoperative treatment was known for 217 of 228 patients, and postoperative locoregional radiotherapy was given to 158 patients. Disease relapse was defined as the first documented evidence of local or regional axillary recurrence or distant metastasis.
Follow-up information was available for 223 patients and included survival status (alive or deceased) and disease status (relapse free or recurrence/metastasis) along with the dates of the events and cause of death, if applicable. The median follow-up was 58 months. The distribution of follow-up times for patients still alive at the time of analysis ranged from 24–74 months with a median of 62 months; only six and two patients had been followed for less than 48 and 36 months, respectively. Follow-up times for the entire cohort, however, ranged from 10–75 months with a median of 58 months. The RFS in each case was the time interval between the date of surgical removal of the primary cancer and the date of the first documented evidence of relapse. The OS was the time interval between the date of surgery and the date of death or the date of last follow-up for those who were alive at the end of the study.
Preparation of Cytosolic Extracts.
Tumor tissues (n = 228) were stored at
-80°C until pulverization in liquid nitrogen and cytosolic
extraction. The extraction procedure consisted of treatment of the
tissue powders (10–50 mg) with a cell lysis buffer (500 µl)
containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5
mM EDTA, 1% NP40, and 1 mM
phenylmethylsulfonyl fluoride for 30 min on ice and subsequent
separation of cell debris from the cytosols by centrifugation at
15,000 x g for 30 min at 4°C. Supernatants
were assayed for total protein concentration immediately after
centrifugation by the Lowry method.
PAP Activity Assay.
The assay measures the incorporation of [5'-8-3H]ATP into
acid-insoluble material using poly(A) as initiator, as described
previously (14
, 18)
. One unit of enzyme activity is
defined as 1 nmol of radioactive radionucleotide incorporated per hour.
Specific activity is expressed as units of activity per milligram of
protein.
Western Blot Analysis.
Cell lysates were run on 8% SDS-PAGE gels and transferred
electrophoretically to Immobilon-P (Millipore) membranes. Rabbit
polyclonal antiserum raised against recombinant bovine PAP expressed in
Escherichia coli (courtesy of Dr. E. Wahle and A. Jenny) was
used at a dilution of 1:2500. Western blots were visualized with the
enhanced chemiluminescence system by Amersham according to the
manufacturer’s instructions. The films were scanned using the UMAX
Scanner (Vista-S6) and quantitated using the Image 1.44 program.
Steroid Hormone Receptor Analyses.
Steroid hormone receptors were quantified as described elsewhere
(21)
. The results of the dual ligand binding assay, in
which dextran-coated charcoal was used to separate bound ligand from
free ligand, were interpreted by Scatchard analysis (22)
.
Tumors with ER and PR concentrations of 
10 fmol/mg protein were
considered receptor negative, those with ER and PR concentrations of
10–300 fmol/mg were characterized as positive, and those with receptor
concentrations above such values were considered strong positive, as
described previously (23
, 24)
.
Statistical Analysis.
For analysis of the data, patients were subdivided into groups on the
basis of different clinical or pathological parameters. Because the
distribution of PAP activity levels was not Gaussian, the analysis of
differences in PAP values between two groups was performed with the
nonparametric Mann-Whitney U test. Similarly, relationships
between more than two groups were determined by the Kruskal-Wallis
test. In this analysis, PAP was used as a continuous variable. PAP
values were also classified into two categories (PAP-positive and
PAP-negative groups), and associations between PAP status and other
qualitative variables were analyzed using the 
2 and
Fisher’s exact tests, where appropriate. An optimal cutoff point equal
to 5.5 units/mg protein was found by 2 analysis. ER and
PR values were categorized into strong positive, positive, and negative
status as described above. Tumor size was classified into three
categories: (a) <2 cm; (b) 2–5 cm; and
(c) > 5 cm. Lymph node status was either
positive (any positive number of nodes) or negative. Age was
categorized into three groups: (a) <45 years;
(b) 45–55 years; and (c) >55 years. Survival
analyses were performed by constructing Kaplan-Meier RFS and OS curves
(25)
, where differences between the curves were evaluated
by the log-rank test and by estimating the RRs for relapse and death
using the Cox proportional hazards regression model (26)
.
Cox regression analyses using SAS statistical software (SAS Institute,
Cary, NC) were used to calculate the RR and 95% CI. Only patients for
whom the status of all variables was known were included in the
multivariate models.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the distribution of the activity, which was slightly positively
skewed. The range of PAP activity in the cytosols derived from 15
nonmalignant samples was 1.5–4.8 units/mg protein; the mean and median
values were 3.5 and 2.8 units/mg protein, respectively. To investigate
the variation in PAP activity values recorded in the breast tumor
cytosols, Western blot analysis was performed to determine the level of
PAP antigen in the cytosols. One major protein band was present in all
tumor cytosols examined, whereas in extracts from the MCF-7 breast
cancer cell line, additional forms of PAP were apparent (Fig. 2)
. Furthermore, the data indicated that PAP activity values correlated
with the level of expression (intensity) of the
Mr 80,000–90,000 protein. Thus, we are
inclined to conclude that the differences in PAP enzymatic activity in
breast tumor cytosols were due to different levels of expression of an
Mr 80,000–90,000 protein rather than
differences in posttranslational modification.
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2 analysis,
based on the ability of PAP values to predict the RFS and OS for the
population studied. As shown in Fig. 3
, the specific activity of 5.5 units/mg protein was shown to be the
optimal cutoff value (2 = 12.7 and
P < 0.001 and 2 = 16.1 and P < 0.001 for RFS and OS,
respectively). This cutoff (47th percentile) identifies
53% of patients as being PAP positive. PAP positivity was found more
frequently in patients with node-positive (P = 0.041) and stage III disease (P = 0.033), in strong positive ER (P < 0.001)
and PR (P = 0.013) tumors, and in invasive
ductal carcinomas. Conversely, PAP positivity was absent in invasive
lobular carcinoma and other histological tumor types
(P < 0.001). No significant associations
between PAP status and patient age, tumor size, grade, and cathepsin D
status were observed (Table 1
; Figs. 4
and 5
).
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. These regression models showed an approximately 2-fold higher risk of
relapse and a 3-fold higher risk of death in patients with PAP-positive
tumors compared with those whose tumors were PAP negative. The
Kaplan-Meier survival curves (Fig. 6)
also showed that PAP-negative patients had more favorable RFS and OS
rates than PAP-positive patients. The difference in survival rates
between the two groups was greater for OS than for RFS. In the
multivariate analysis of PAP, the Cox regression models were adjusted
for age, nodal status, tumor size, grade, and cathepsin D, ER, and PR
status, which were used as categorical variables (except for tumor
size, which was used as a continuous variable) as described above.
Patient age, tumor size, and ER and nodal status were thus shown to be
independent factors for predicting both RFS and OS. PAP significantly
added to the prognostic power in the multivariate model of RFS
(RR = 2.51; P = 0.0012) and OS
(RR = 4.21; P < 0.001)
analyses.
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and Fig. 7
. Node-negative patients with PAP-positive tumors tended to have an
approximately 5-fold increase in risk for relapse or death. PAP
activity proved to be an independent factor for predicting RFS
(RR = 3.68; P = 0.0032) and OS
(RR = 4.81; P < 0.001) in
node-negative patients (Fig. 7, A and B)
.
Cathepsin D and ER significantly added to the prognostic power in the
multivariate model of analysis for RFS and OS, respectively. In
node-positive patients, no statistically significant difference was
observed in RFS and OS between PAP-positive and -negative tumors (Fig. 7, B and D)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, PAP activity was measured in the cytosols of 228 women, and these values were analyzed comparatively with the follow-up data of 223 of the patients. On dividing the patients into PAP-positive and PAP-negative groups using a cutoff value of 5.5 units/mg protein, a relation emerged between PAP activity and other unfavorable prognostic parameters. A higher percentage of tumors positive for PAP activity was recorded within the node-positive group of patients, the stage III disease group, and the invasive ductal tumors, in comparison with other histological types. These data confirmed previous observations linking high PAP activity levels with unfavorable prognostic parameters in breast cancer.
This is the first study of the prognostic value of PAP activity in breast cancer. Follow-up information for 223 patients indicated that patients with PAP-positive tumors had a 2-fold increase in risk of relapse and a 3-fold increase in risk of death, as compared with those with PAP-negative tumors. PAP activity significantly added to the prognostic power of parameters such as age, nodal status, tumor size, grade, and cathepsin D and steroid receptor status in the multivariate models of analysis for both RFS and OS. The prognostic significance of PAP activity was more prominent within the group of node-negative patients. A 5-fold increase in the risk for relapse or death was calculated for node-negative patients whose tumors showed high PAP activity. Our data indicate that PAP activity may be an independent factor of poor prognosis.
Because very little is known about the physiological role of PAP in breast tissue, a hypothesis explaining the mechanism by which PAP expression may confer poor prognosis in breast cancer, especially in node-negative patients, is at present difficult to formulate; it is also difficult to correlate PAP measurements with global or individual mRNA polyadenylation, a highly regulated multicomponent process. A strong positive association between high ER content (>300 fmol/mg) and PAP activity was observed. The presence of ER in tumor cells is a well-established predictor of response to endocrine therapy but is a weak prognostic indicator. Moreover, a very high ER concentration may be a negative prognostic indicator. Patients with very high ER content have been reported to have a poor prognosis compared with patients with a low concentration of ER (29 , 30) . It was recently reported that whereas the presence of ER and expression of the proliferation-related marker Ki-67 are mutually exclusive in normal proliferating epithelium, in breast cancer, they are often coexpressed (31) . If PAP is increased in proliferating cells, it is not surprising that its expression correlates with ER content.
The poly(A) tail is present at the 3' end of virtually all mRNAs and affects both cytoplasmic mRNA stability and translatability (12) . Formation of this structure involves endonucleolytic cleavage of the mRNA precursor coupled with poly(A) synthesis, a reaction that requires several protein factors. Two multisubunit factors are required for specification of the poly(A) site and formation of a stable protein-RNA complex. Subsequently, two additional proteins, as well as PAP, join the complex and are required for cleavage of the pre-mRNA and synthesis of the poly(A) tail (32) . Regulation of individual components of the polyadenylation process has been established, for example, during the cell cycle and cellular differentiation (13 , 33 , 34) . Furthermore, inappropriate RNA processing is prevented by the BRCA1-associated RING domain protein (BARD1) that interacts with the polyadenylation factor CstF-50 and inhibits polyadenylation in vitro (35) .
In mammalian cells, high levels of PAP activity have been reported in rapidly proliferating cells and in neoplastic cells (11 , 14, 15, 16, 17 , 36) . Earlier reports attribute increased levels of PAP activity in neoplasia to phosphorylation (37) . However, recent data indicate that among the several species of PAP characterized by varying degrees of phosphorylation, the hyperphosphorylated species represents a form with reduced enzymatic activity. It has been shown that PAP can be phosphorylated in vivo and in vitro by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor) at four nearby nonconsensus cyclin-dependent kinase sites (38) . PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell cycle at which maturation/mitosis-promoting factor is known to be active (13 , 39) . Reduced PAP activity probably contributes tot he well-established reduction in polyadenylated mRNA and/or protein synthesis known to occur in M-phase cells (13 , 38) . In the breast cytosols studied, only one species of PAP was detected by the polyclonal antibody used, and the intensity of the band corresponded to the enzymatic activity measured in each sample. The same antibody recognized additional species in MCF-7 extracts and multiple forms of PAP in other cell lines (40) . The possibility that additional forms of the enzyme exist in the intact breast tumor cells cannot be excluded. However, the above-mentioned data confirm that the high activity measured in certain samples reflects overexpression of the enzyme.
In breast cancer, the degree of lymph node invasion is one of the major prognostic factors for predicting relapse and death (3 , 41 , 42) . On the other hand, about 30% of node-negative tumors relapse, and it has been proposed to apply systematic adjuvant chemotherapy to all patients, including those who are node-negative (43) . However, this adjuvant treatment may be detrimental to patients who do not require it. In our study, PAP-positive status in node-negative patients is not only an adverse prognostic factor, but PAP-positive patients in this group have substantially worse prognosis than node-positive patients, regardless of PAP status. This may suggest the potential utilization of PAP to predict which of the node-negative patients should be treated.
The level of PAP activity in the cytosols of breast tumors may be a parameter related to proliferation and/or transcriptional activity of the tumor cell. Alternatively, high levels of PAP activity may reflect deregulated expression of this protein, which in turn may contribute to the malignant phenotype of the cell. Both possibilities merit further examination. Nevertheless, our data indicate that high levels of PAP activity characterize a more aggressive tumor type, and PAP has been proven to be a new independent prognostic indicator, particularly in node-negative patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Greek Ministry of Health,
European Union Training for Mobility of Researchers. Grant ERBFM
RXCT-960096 (to C. M. T.), and General Secretariat of
Research and Technology Grant 9427/13.9.96 (to T. T.). 
2 Present address: Department of Pathology and
Laboratory Medicine, Mount Sinai Hospital and Department of Laboratory
Medicine and Pathobiology, University of Toronto, 600 University
Avenue, Toronto, Ontario, M5G 1X5 Canada.
3 To whom requests for reprints should be
addressed, at Papanikolaou Research Center, St. Savvas Hospital, 171
Alexandras Avenue, Athens 11522, Greece. Phone: 30-1-6409168; Fax:
30-1-6420146; E-mail: T.Trangas{at}genet.ath.forthnet.gr
4 The abbreviations used are: OS, overall
survival; PAP, polyadenylate polymerase; RFS, relapse-free survival;
ER, estrogen receptor; RR, relative risk; poly(A), polyadenylate;
PR, progesterone receptor; CI, confidence interval.
Received 11/17/99. Accepted 8/ 2/00.
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REFERENCES |
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