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Molecular Oncology, Markers, Clinical Correlates |
Research Laboratory for Reproductive Health, Department of Woman and Child Health, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm [M. G., Å. P.], and the Departments of Obstetrics and Gynecology [M. G., K. C.], Pathology [B. L. R.], Urology [R. S.], and Clinical Research Center [K. C.], Karolinska Institutet, Huddinge University Hospital, S-14186 Huddinge, Sweden
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Statistical evaluation showed that the T-PSA ratio between month 12 and month 0 had the most significant prognostic value for predicting the clinical outcome. This ratio was superior to clinical classifications, e.g., tumor stage and cytological grade, and also was higher than T-PSA at the time of diagnosis. This study has shown that aspiration biopsy material can be used to reveal biochemical changes in the tissue during treatment and that one specific marker (T-PSA) can predict the clinical outcome of endocrine treatment of CaP patients better than previously used methods. We believe that selected tissue markers or the protein pattern can help us to characterize the tumors and predict the clinical outcome so an optimal treatment can be chosen for every patient.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S-PSA is widely used for monitoring treatment and also to some extent for diagnosis of CaP (1, 2, 3) . After radical prostatectomy, increasing S-PSA values indicate residual disease and recurrence of tumor (4 , 5) . This is also true for endocrine treatment of CaP, including M1 patients. After initiation of treatment, S-PSA usually drops to normal and increases subsequently when the tumor becomes hormone insensitive (6) . The prognostic value of total and free PSA has been discussed by many research groups (3 , 7, 8, 9) . Nevertheless, the usefulness of S-PSA analysis has not drastically improved in this respect.
In experimental studies we found lower PSA values in a cell line that was hormone resistant (LNCaP-r) compared with a hormone-sensitive cell line (LNCaP). On the basis of these results, we developed a method for quantitation of PSA in fine-needle aspiration biopsies from patients with prostate diseases (T-PSA). T-PSA was shown to correlate negatively to clinical stage and cytological grade, that is, highly malignant tumors have low tissue concentrations of PSA (10, 11, 12) . We also found an inverse correlation between pretreatment PSA concentrations in tumor tissue and in serum from patients with prostate cancer (13) . In a 2-year follow-up study of hormonally treated CaP patients, we showed that T-PSA appeared to be a valuable adjunct to cytological grading for predicting progressive disease (12) . In a recent study we retrospectively correlated pretreatment variables to the clinical outcome in 179 patients and showed that T-PSA was the single most important factor to predict time-to-progress and time-to-disease-specific death (14) .
The major aim of this study was to reveal the effects of different hormonal treatments on PSA concentration in the prostate. The second aim was to evaluate retrospectively the prognostic value of serial T-PSA determinations during endocrine treatment of patients with CaP.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The patients were in a nonrandomized procedure allocated to three forms of hormonal treatment pending exclusively on patients decision. The following treatments were given: surgical castration by bilateral orchidectomy (30 patients) or medical castration with GnRH agonists [3.6 mg goserelin acetate (Zoladex; Zeneca, Wilmington, DE) s.c. every fourth week; 11 patients] or with parenteral depot estrogens [240 mg polyestradiol phosphate (Estradurin; Wyeth-Ayerst, Philadelphia, PA) i.m. per month; 22 patients]. Both regimens of medical treatment suppress circulating testosterone to castration levels (15 , 16) .
Follow-Up and Survival.
The patients were included in the study from August 1986 until March
1988 and were recorded until April 1997. They were followed-up and
evaluated according to the recommendations of European Organization for
Research on Treatment of Cancer. Clinical examinations were performed
every 12 weeks, including assessment of prostate dimension by digital
rectal examination and control of location and dimension of soft tissue
metastases. Bone scan and/or conventional X-ray for assessment of bone
metastases was performed every 24 weeks during the entire observation
period. Objective progression of the disease was defined as an increase
of T stage by two steps or more compared with the lowest T stage
earlier recorded. The appearance of skeletal or nonskeletal metastases
also was recorded as objective progression. All of the patients were
followed-up until the end of the observation period or until death.
Aspiration biopsies were taken before (0) and at 6, 12 and 24 months
after diagnosis.
When this study was initiated, S-PSA assays were not routinely used in our departments. A comparison between S-PSA and T-PSA therefore was not possible in this study.
T Staging.
T staging was performed exclusively by digital rectal examination
throughout the entire period of investigation by the senior urologist
(R.S.), according to Union International Contre Cancer (UICC)
guidelines (17)
. Stages T1 and
T2 are confined to the gland and correspond to
stages A and B, respectively. In stage T1 the
tumor is surrounded by palpably normal gland. Stage
T2 has a smooth nodal deforming contour, without
involvement of lateral sulci and seminal vesicles. Stages
T3 and T4 are tumors
extending beyond the capsule, corresponding to stage C. In stage
T3 the tumor extends beyond the capsule with or
without involvement of lateral sulci and/or seminal vesicles. In stages
T4 the tumor is fixed or infiltrates neighboring
structures. Ultrasound equipment for transrectal examination of the
prostate was not available at our department when this study was
initiated.
Fine-Needle Aspiration Biopsies.
Fine-needle aspiration biopsies were obtained during routine
examination according to the method of Franzén (18)
.
All of the biopsies were taken by the same cytologist (B.L.R.). Four
biopsies were obtained from the same tumor area. Two biopsies were
prepared for morphological analyses and two for determination of T-PSA.
To be able to perform the planned aspiration biopsies in the same tumor
area, the pathologist did a meticulous depiction of the primary tumor
location for each patient.
Cytology.
Cytological grading of the aspirates was performed through the entire
period of investigation by the same senior pathologist (B. L. R.)
without knowledge of the biochemical data. Three grades of malignancy
were defined based on six cellular properties: average nuclear size,
average nucleolar size, variability in nuclear size, disturbance of
nuclear arrangement, and cellular/nuclear dissociation
(19)
.
Biochemical Analyses.
Samples were stored at -70°C until analysis. Cytosols were prepared
by sonication of the biopsies followed by centrifugation at
105,000 x g. During these procedures all of the
samples were kept on ice or refrigerated. Cytosolic PSA was determined
by RIA and DNA content in the biopsy material by fluorometry
(10)
. The tissue content of PSA in the aspirates is given
as µg PSA/µg of DNA.
Statistical Methods.
Differences between groups were tested by Mann-Whitney U
test and changes in T-PSA during treatment by ANOVA for repeated
measurements. In the survival analyses the 11 M1
patients known to have an adverse prognosis were excluded. The
categorical factors, T-stage and cytological grade, were analyzed
separately in a Cox univariate analysis. To evaluate the possible
prognostic value of the change in T-PSA in 52 M0
disease patients, we started the survival analyses when PSA was
measured the second, third, and fourth time after diagnosis. The
changes in T-PSA values were expressed as a ratio between T-PSA at 6,
12, and 24 months compared with the initial T-PSA values (T-PSA at
diagnosis). Cox univariate analysis initially was carried out for these
three different ratios and also the initial T-PSA. The same subset of
prognostic variables, including the above mentioned categorical
factors, was selected for stepwise Cox bivariate models to evaluate the
most useful predictor (20
, 21)
. Data are presented as
arithmetic means ± SE or as median and range according to
distribution.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Tumors with a high malignancy grade
were more frequently in stage T4. Fifty-two
patients had M0 and 11 M1
disease at the time of diagnosis.
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. T- PSA values decreased with
increasing tumor stage and cytological grade, except for the
T2 stage tumors which was attributable to small
sample size (3 patients). T-PSA levels also decreased significantly in
the patients with metastatic disease.
Follow-up and Survival.
For the patients who survived (n = 14) the median
follow-up timewas 111.5 (range, 98–128 months). Corresponding values
for the patients who died of CaP (n = 31) and the
patients who died of causes other than CaP (n = 18)
were 28.9 6–70(6–70) and 48.3 months (18–109 months), respectively.
For analysis of outcome, patients were divided into two groups according to the metastatic situation at time of diagnosis. Twenty-one of the 52 M0 patients died of CaP, and 31 were alive at the end of the observation period or died of causes other than CaP (14 and 17 patients, respectively). Ten of the M1 patients died of CaP, and 1 patient for other reasons than CaP.
T-PSA Values before and during Treatment in Relation to Outcome.
Figure 1
and Table 2
show T-PSA analyzed before and during
hormonal treatment for all of the 63 patients. Group A represents
patients who were alive at the end of observation period or died of
causes other than CaP. Group B includes patients who died of CaP.
Pretreatment values of T-PSA (time of sampling, 0 months) were
significantly higher in group A compared with patients in group B
[0.381 (0.050–20.911) versus 0.063 (0.002–0.723) µg
PSA/µg DNA, P < 0.001]. On the other hand, there
was no difference in pretreatment T-PSA values between
M0 and M1 patients who died
of CaP [0.067 (0.010–0.723) versus 0.043 (0.002–0.231)
µg PSA/µg DNA, P > 0.3]. In group A the T-PSA
values decreased significantly during treatment (P =
0.023). In contrast, T-PSA values of group B patients increased
successively during treatment (P < 0.001).
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and 4
.
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. Seventeen had been
treated by orchidectomy or GnRH agonists and 14 with estrogens. Because
surgical castration by bilateral orchidectomy and medical castration
with GnRH agonists produce similar sex steroid conditions, these groups
were combined in the statistical calculations. T-PSA values decreased
significantly from pretreatment values and stayed at lower values in
the orchidectomized/GnRH agonist–treated group (P =
0.031). In contrast, the T-PSA decrease in the group receiving
estrogens showed no significance (P = 0.248). There was
no difference in pretreatment T-PSA values between the two treatment
groups (P > 0.4). In contrast, values during treatment
were significantly higher in the patients treated with estrogens
compared with the other group (0.001 
P 
0.005).
Group B.
In this group 15 patients had been treated by bilateral orchidectomy or
GnRH agonists and six patients with estrogens. All of the patients
showed increasing T-PSA values during treatment. In the group treated
with surgical castration or GnRH agonists the increase was significant
(P = 0.002). Pretreatment values of T-PSA were
significantly higher in patients receiving estrogen therapy
(P < 0.005), but there was no influence of this
treatment regimen on T-PSA values during treatment in group B (0.4 P 0.9).
In detail, at 6 months we observed a definite increase in T-PSA values
in 11 patients. Only 2 of them developed clinical progression within
this 6-month period (Table 5)
. The other
9 patients developed a later clinical progression (median, 26 months).
After 12 months, T-PSA values had increased in an additional 9
patients. Six of these patients at this time had no signs of clinical
progression, which was first observed later (median, 22 months). One
patient showed clinical progression after 4 months. In this case a
definite T-PSA increase was seen first after 24 months (Table 5)
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The statistical calculations using Cox univariate analysis of
M0 patients with respect to disease-specific
death are shown in Tables 6
and 7
. The change between the T-PSA values at
12 months and the initial values is the best predictive factor. Other
competing factors of less importance were T-PSA value changes at 6 and
24 months, T-stage, and cytological grade. Also a Cox bivariate
analysis confirmed that the T-PSA ratio between 12 and 0 months is the
best predictor for outcome of the disease. T-stage and cytological
grade were nonsignificant competing factors (Table 8)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For these studies it is of vital importance that the aspiration procedure is highly standardized and performed by an experienced cytologist. Using the fine-needle aspiration method for biopsies makes it possible to obtain tissue material from a circumscribed area of the gland a number of times during the treatment. The material is also rather specific, because studies by Tribukait et al. (22) showed that an average of 75% of all of the cells in fine-needle aspirates of prostate carcinomas are tumor cells. In well and poorly differentiated tumors, the average values were 66% and 85%, respectively, and the mean incidence of inflammatory cells was 3%.
A number of other research groups have studied intracellular markers, e.g., PSA, and correlated these results to the histological findings (23) . Many of these studies are performed on tissue material obtained from operation specimens or core biopsies, which almost always contains a high degree of different components of prostate tissue. If therefore significant proportions of normal prostate cells are present in the material, the measured PSA values are overestimated. On the other hand, true values are underestimated if a significant proportion of stromal or inflammatory cells are present in the materials.
Our major goal was to reveal the effect of different endocrine treatment on T-PSA, and an interesting finding was that T-PSA levels during treatment in patients who responded to endocrine therapy were significantly higher in patients treated with estrogens than in those treated with orchidectomy/GnRH agonists and that estrogen treatment did not result in any pronounced decrease in T-PSA levels. The synthesis of PSA is considered to be androgen sensitive, and androgen depletion is known to decrease S-PSA levels in patients with hormone-sensitive CaP. The regulation of PSA, however, is complex, and other factors may affect both the synthesis and secretion. In vitro, besides androgens, estrogens also have been shown to stimulate PSA synthesis in LNCaP cells (24 , 25) . Our finding indicates that estrogens also may have this ability in vivo. The effect of estrogens also is of interest because there are indications for a role of estrogens in the etiology of CaP (Ref. 26 and references cited therein). Besides a hypothetic stimulatory effect of estrogens on cellular PSA synthesis, there also may be the possibility that estrogen treatment affects T-PSA levels by changing the relation between epithelium and stroma. However, investigation of such a relationship implies the microdissection technique, which was not available at our department at the time of our investigation.
In the patients who did not respond to endocrine therapy, the initial T-PSA levels were significantly lower than in the patients who responded to therapy but increased dramatically during treatment. Also, there were no differences related to therapy regimen in T-PSA levels during treatment in these patients. These findings clearly illustrate that other factors are responsible for the regulation of T-PSA levels in these tumors. One possibility is that the PSA synthesis has become androgen insensitive, but another possibility is that gene amplification of the androgen receptor occurred and that the adrenals stimulate PSA synthesis.
In previous studies we could relate a decrease in T-PSA values to an increase in malignancy grade and tumor stage (11) , and we also have shown that T-PSA at the time of diagnosis is the best single prognostic factor when compared with serum total and free PSA, free-to-total serum PSA ratio, tumor stage, cytological grade, and DNA ploidy to predict the clinical outcome (12 , 14) . These results are confirmed in this study, and an even higher prediction could be found using the 12- to 0-month T-PSA ratio, which can be considered as a biochemical analysis of the changes that occurred in the tissue.
Compared with serum PSA, T-PSA is less dependent on factors not related to the production of protein in the tissue, such as volume of the PSA-producing tissue, transport of PSA into the blood, and clearance of the protein from the circulation. Any process that alters any of these factors will affect S-PSA levels (27) . Therefore, T-PSA reflects the biochemical composition of the gland more physiologically, e.g., T-PSA levels are high in normal tissue and in hyperplastic prostate tissue (10) .
We also observed that pretreatment T-PSA concentrations in patients with M0 or M1 disease at time of diagnosis are not different in the group of patients who died of CaP. This indicates that the factors involved in metastasizing do not effect the PSA concentration.
We initially found it strange that one single biochemical tissue marker can predict so well the clinical outcome. However, we consider today T-PSA as a marker for normal prostatic tissue, and a decrease should be considered as sign of abnormal regulation. In line with this, we believe that it is possible to develop these methods further and to use the aspiration biopsy material to analyze other markers of interest or characterize the protein pattern by two-dimensional gel techniques. By doing this, we could learn much more about the biochemical changes that occur during the development of CaP at the same time, because we can explore the use of different markers for predicting the clinical outcome. In this way we also should be able to select the optimal treatment, which means both avoiding treatment when not necessary and treating aggressively when not necessary.
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FOOTNOTES |
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1 Supported by grants from the Swedish Medical
Research Council (11615), Karolinska Institutets Fonder, and Leo
Research Foundation. 
2 To whom requests for reprints should be
addressed, at Research Laboratory for Reproductive Health, C4 U1,
Department of Woman and Child Health, Karolinska Hospital, S-171 76
Stockholm, Sweden. Phone: 468-51772403; Fax: +468-323048; E-mail: ake.pousette{at}kbh.ki.se
3 The abbreviations used are: CaP, carcinoma of
the prostate; PSA, prostate-specific antigen; S-PSA, serum
concentrations of PSA; T-PSA, PSA in the tissue; GnRH,
gonadotropin-releasing hormone.
Received 10/ 5/99; revised 2/ 7/00; accepted 2/14/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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