Prostate cancer is the most commonly diagnosed noncutaneous cancer in men in the Western world and is the second most frequent cause of cancer-related deaths in men.1 More than 95% of malignant prostate neoplasms are adenocarcinomas, and in general, the term prostate cancer is used to designate primary adenocarcinoma. However, prostate cancer comprises a wide spectrum of biological and clinical behavior, ranging from a minute latent tumor to a highly aggressive, life-threatening disease.2 Today, majority of prostate cancers are detected based on serum prostate-specific antigen (PSA). The ultimate goal of prostate cancer screening is to reduce the disease-specific mortality. However, because the biological behavior and the natural course of prostate cancer and its relation to PSA is not known, controlled randomized screening studies are needed to evaluate the effects of early detection and treatment of cancer in mortality and in quality of life.3 The European Randomized Study of Screening for Prostate Cancer (ERSPC) is a multicenter, randomized trial, which started in 1994.4 Within the ERSPC, approximately 193,000 men from 8 European countries have been randomized, of whom approximately 40% are from Finland with a sample size of approximately 80,000 men.5
Pathological stage and tumor grade together with PSA are the most important indicators of outcome.6 However, additional markers for growth potential are needed, because of variation of growth potential and prognosis among tumors with similar stage or grade. The proliferation marker Ki-67 (also known as MIB-1) is a protein that recognizes nuclear antigens in the cell cycle from the late G1 phase, in S and G2 phases to M phase but not in noncycling cells in G0 phase.7 Ki-67 expression is associated with the prognosis of many malignant tumors, such as breast cancer,8 colorectal adenocarcinoma,9 and also prostate carcinoma.10 Ki-67 expression is associated with pretreatment prostate-specific antigen (PSA) level, tumor grade (Gleason score), pathologic stage and progression of cancer.11, 12 Patients with higher Ki-67 staining have worse prognosis than those with a lower expression.13 We have recently shown that Ki-67 staining is an independent prognostic marker in prostatectomy treated patients.14
The aim of this study was to characterize prostate cancers diagnosed during the first 2 rounds of the screening trial and compare their tumor characteristics with other cancers in the trial population, including interval cancers and cancers among nonparticipants. The characterization was performed from the diagnostic biopsies and the indicators of biologic aggressiveness were Gleason score, focality or bilaterality of tumor and Ki-67 expression.
Material and methods
The Finnish prostate cancer screening trial is the largest component of the ERSPC with a cohort of 80,458 men aged 55–67 years at entry. The study population was identified from the Population Register of Finland, and those men with previously diagnosed prostate cancer were excluded before randomization (n = 1,570). Annually, during 1996–1999, 8000 men aged 55, 59, 63 and 67 years were randomized into the screening arm, and the remaining men formed the control arm. At the time of invitation, 30,403 men were eligible and finally, 20,793 participated in the first and 19,107 men participated in the second screening round after 4years.
For the present study, we randomly selected 128 cancers out of a total of 543 screen-detected cases from the first round. Of these 126 (98%) histologic samples were available. Of the 508 screen-detected cases in the second round, 125 cancers were selected and 123 samples were available. During 1996–2003, a total of 863 cancer were diagnosed in the control arm and a random sample of 135 cancers was selected for the study (133 were available). All the prostate cancers diagnosed among nonparticipants in screening (92 cancers, of which 89 histologic samples were available) and the cancers diagnosed between the screening rounds (106 cancers, of which 99 samples were available) were included in the study. The cancers were defined as interval cancers, if they were diagnosed during the screening interval following either normal PSA or elevated PSA, but negative biopsies. The cancers diagnosed in the control arm and among nonparticipants were identified from the Finnish Cancer Registry.
Men with serum PSA level 4.0 ng/ml or higher were referred for diagnostic examination [a digital rectal examination (DRE), a transrectal ultrasound (TRUS) and sextant needle biopsy with 18 G needles]. Additional biopsies could be taken from suspicious nodules. For men with PSA of 3.0–3.9 ng/ml, an ancillary screening test was performed (DRE in 1996–1998 and determination of the proportion of free PSA with a cut-off value of 0.16 since 1999).
If the first biopsy indicated a cancer suspicion or high-grade prostatic intraepithelial neoplasia or if the material was insufficient, 1 repeat biopsy was suggested. If the serum PSA was above or equal to 10 ng/ml, a repeat biopsy was also recommended regardless of the benign histological finding in the first biopsy.
The histologic samples were available from 570 prostate cancers, of which 540 were biopsies and 30 were resection materials. Material for the first and the second screening rounds was classic sextant biopsy cores throughout the study. The samples were fixed in buffered formalin and embedded into paraffin blocks according to routine protocol. Each slide was stained with hematoxylin and eosin. The primary cancer diagnosis was made by general pathologists in several laboratories. All the samples with a primary diagnosis of prostate cancer were reviewed and the diagnoses were confirmed by a single uropathologist (M. Laurila). All the tumors were regraded according to the Gleason system. Tumors were classified in 3 groups: cancers with Gleason score <7, score 7 and score >7. The number of the needle biopsy cores and the number of the cores containing cancer were counted. Focal cancers, with a malignant focus of maximum 3 mm and a Gleason score of maximum 6 in a single biopsy core, were reported. The cancers in biopsies from both prostate lobes were reported as bilateral cancers.
The block with the most representative slide containing the largest tumor volume from each case was selected for the immunohistochemical staining. The volume of cancer in biopsy cores was small in some cases, especially in focal cancers, and therefore, no cancer tissue was available for immunostaining in 91 samples. The proliferation antigen Ki-67 (MIB-1) could be analyzed in 479 cases (84%) of 570 cancers.
A 5-micron section was cut from the representative block of formalin-fixed, paraffin-embedded tissue and placed on silinated slides. For the antibody, the sections were deparaffinized in graded alcohols and immunostained with the monoclonal antibodies to Ki-67 antigen (MIB-1, clone MM-1, Novocastra Laboratories, Newcastle, UK) according to manufacturer's instructions. High-temperature antigen retrieval was performed with 20-min incubation in 1 mM ethylenediaminetetra-acetic acid, pH 8.5. The bound antibody was visualized using a standard avidin-biotin technique (Vectastain Elite, Vector Laboratories, Burlingame, CA).
The Ki-67 stainings were graded by a single uropathologist (M. Laurila). Most of the specimens showed a heterogeneous distribution of immunostaining. For Ki-67 staining interpretation, brown, granular nuclear reactivity was considered positive. Tumor areas showing the highest density of cells were chosen for analysis. Positively stained tumor nuclei were calculated under 400-fold magnification. The whole tumor, or at least 5 areas were selected, and a minimum of 1,000 tumor cells were scored. The mean percentage of positive tumor nuclei per counted tumor cells was characterized as low (occasional or up to 5 percent positive cells), moderate (5 to 10 percent positive cells) or high (more than 10 percent positive cells).
Comparisons of proportions of focal and bilateral cancers, Gleason score and proliferation antigen Ki-67 between patient groups, as well as comparison of proliferation with Gleason score were done by Pearson χ2 test. Logistic regression models with odds ratios (OR) and 95% confidence intervals (CI) were used to examine the association of focal and bilateral cancers in 5 prostate cancer subgroups. Statistical analyses were performed using SPSS for Windows, version 14.0.2. A p-value less than 0.05 was considered statistically significant.
The total number of prostate cancers in the 5 subgroups (the first round, second round, nonparticipant, interval cancers and control arm), including focal and bilateral cancers in each subgroup, are presented in Table I. Of all 570 cancers 82 (14%) were focal tumors. Most of them (89%) were Gleason score 6 tumors; only 2 focal cancers were graded as Gleason score 4 and 7 as Gleason score 5. The proportion of focal tumors differed significantly between the subgroups (p = 0.04). In the second round, the proportion of focal cancers was 2.8 times higher than in cases detected than in the control arm. Focal cancers were also more than twice as common in the interval cases compared to the control group OR 2.63 [95% CI 1.19–5.23].
Table I. Distribution, Number of Cases (n) with Percentages (%), of Focal and Bilateral Cancers in Different Subgroups (n = 570)
Differences between subgroups were tested by Pearson χ2 test.
p = 0.039
p < 0.001
Of the cancers, 157 (28%) were bilateral and the proportion of bilateral cancers differed significantly between the groups (Table I). In the first and second round of screening as well as interval cancers significantly fewer bilateral cancers were detected than in the controls and nonparticipants (p < 0.001). In the control arm, the proportion of bilateral cancers was 2-fold higher (OR 2.88 [95% CI 1.66–5.01]) compared to the first round and second screening round (OR 2.67 [95% CI 1.54–4.61]), and nearly 3-fold compared to the interval cases (OR 4.20 [95% CI 2.20–8.03]).
More than half of the tumors diagnosed in the first round were high-grade cancers (Gleason 7 or higher) (Fig. 1a). In the second round, the proportion of high grade cancers decreased and low-grade tumors increased from 47% to 67%. The Gleason score in interval cancers was intermediate between the first and the second round. The highest Gleason scores were in the nonparticipants and in the control arm. Bilateral cancers had high Gleason score in every subgroup (Fig. 1b, p = 0.002).
The proliferation antigen Ki-67 (MIB-1) could be analyzed in 479 (84%) of 570 cancers (from 78% of nonparticipants' to 90% of first round cancers). Ki-67 was associated with the Gleason score: tumors with a low Gleason score had a low degree of Ki-67 expression, and correspondingly, tumors with a high Gleason score showed most frequently high Ki-67 staining (Fig. 2). Ki-67 could be analyzed in 46 (56%) of 82 focal cancers and in 135 (86%) of 157 bilateral cancers. Ki-67 distribution differed significantly between the subgroups in total material (p = 0.039, Fig. 3a). Focal cancers generally expressed Ki-67 at low level, whereas more than half of the bilateral cancers expressed Ki-67 ≥5%. Ten focal cancers of 46 (22%) had proliferation index more than 10%. Ki-67 expression was most common in cancers among nonparticipants and in the control group (Fig. 3). Cancers diagnosed in the second round were positive for Ki-67 slightly more commonly than those detected in the first round. The interval cancers had the lowest Ki-67 expression.
Our material of 249 randomly selected cancers diagnosed in the first and the second round represented approximately 1 quarter of the total number of cancer cases in each screening round, and 40 focal cancers of the screen detected cases were found. This should correspond to 160 focal cancers, found in the first and in the second screening rounds, with the given sampling ratio. The total number of focal cancers in the screening would thus be 192 including the 20 focal cancers in the interval cases and the 12 in the nonparticipants. In the control arm 10 cancers of 133 randomized cases were focal cancers. Based on a sampling ratio of 15%, a total of 67 focal cancers in the whole control arm could be estimated. The cumulative incidence of the focal cancer was 6.3 per 1,000 (192 focal cancers in 30,430 men in screening arm) and 1.4 per 1,000 men in the control arm (67 focal cancers of 48,000 controls) corresponding to a rate ratio of 4.5 (95% CI 3.4–5.9). For the bilateral cancers, the detection rates were 8.6 per 1,000 men in screening arm (n = 262) and 7.9 per 1,000 men in the control arm (n = 378). This corresponds to a rate ratio of 1.1 (95% CI 0.9–1.3).
We found that in a population-based screening program, the biological aggressiveness of the cancers diagnosed in the second screening round was lower than in the first round or that of the interval cancers, while the nonparticipants had the most aggressive cancers. Two possible explanations could account for the findings: either early detection results in diagnosis of cancers before they develop more malignant features or, alternatively, the lower degree of aggressiveness is due to detection of indolent tumors. Both explanations could account for the more aggressive features in the control group and nonparticipants. The low aggressiveness of interval cancers suggests that a large proportion of them was not true de novo cancers arising during the screening interval, but commonly detected after repeated biopsy rounds in screen-positive men.
Screening is aimed at reducing mortality by detecting cancers in their early, asymptomatic phase, while they are still curable. Because of the wide spectrum of clinical behavior in prostate cancer, it is crucial to distinguish cancers that would progress from those that would remain indolent. In our earlier study, we have shown that biological aggressiveness of screen detected cancers is intermediate between clinically detected cancers and latent cancers verified in autopsy.15 However, the results of present study suggest that a proportion of screen-detected cancers are clinically significant. This poses a major challenge as we should know whether the cancer is a relatively harmless and does not require treatment, manageable by active surveillance or watchful waiting, or potentially aggressive demanding more radical treatment modalities.
A meaningful proportion of screen-detected cancers are minimal and could remain indolent during the rest of the man's lifetime without progressing into clinical cancer. It is, however, difficult to predict the natural course of a cancer due to uncertainties in estimating both life expectancy and prognosis of the tumor. Our primary results were given as proportions of cancer in each group fulfilling criteria for aggressiveness such as Gleason score, MIB-1 staining, etc. These results reflect the relative frequencies of cancers with varying aggressiveness. As the screen-detected cancers represent a mixture of indolent and clinically relevant cancers, the former dilute the aggressiveness of those cancers that would be relevant for mortality. To overcome this limitation, we also give the findings as cumulative incidence. Our results indicate a rate ratio of 4.5 for focal cancer, i.e., screening increases the detection of minimal tumors. This gives a crude estimate of overdiagnosis. However, due to leadtime (anticipation on earlier detection of tumors by screening, estimated as 6–12 years for prostate cancer) the control group is likely to catch up with further follow-up to some extent and the difference is therefore likely to decrease. In particular, the expected effect of screening is an eventual reversal of the ratio between the trial arms in the occurrence of clinically progressive tumors (such as bilateral cancers), because effective screening should decrease the risk of cancer with poor outcome.
Only in a randomized trial, the characteristics of screen-detected cancers can be compared with control cancers in a valid fashion, because randomization creates comparable groups and the control arm represents the situation in a similar population in the absence of screening. Volume of prostate cancer in biopsies correlates with the volume of prostate cancer in radical prostatectomy, and 12 biopsies have better predictive value compared to 6 biopsies.16 However, tools to predict prostate cancer characteristics and extent are still limited, and it is uncertain, how volume and biopsy characteristics predict the clinical behavior of the cancer. In prostate biopsy, a small single focus of cancer with a low Gleason grade suggests probably clinically insignificant, organ-defined cancer with low tumor volume. Different definitions for small cancers in biopsy have been used. In the present study, we used the definition of Weldon et al.17 to define focal cancer, i.e., as a single cancer positive biopsy core with a small focus, 3 mm or less, of adenocarcinoma without high grade cancer (Gleason grade 4 or 5). This definition has also been used in the analysis of biopsy results from Dutch screening centre of ERSPC.18 By this definition, our results showed that focal cancer is more frequent in screen-detected cancers than in cancers among controls or nonattendants. Furthermore, the proportion of focal cancers increases further at the second screening round. These results are in accordance with those reported by the Dutch group18, 19 as well as the general ERSPC experience.20 In the study by Postma and others, the proportion of focal cancer increased significantly from 16% in the first round to 29% in the second screening round. This finding is similar to the results of Swedish section of ERSPC in subsequent screening rounds at a 2-year interval.21 Also, in our study the proportion of focal cancer increased from 12% in the first round to 20% in the second screening round. Interestingly, the interval cancers had similar frequency of focal cancers as detected in the second screening round. This indicates that few aggressive cancers develop during the screening interval.
At present we do not yet know, what the real outcome of these focal cancers is. In earlier studies,22 focal cancer in biopsy predicted minimal cancer (less than 0.5 ml) correctly in 53% of subsequent radical prostatectomy specimens. Stamey et al. suggested that tumors smaller than 0.5 ml, regardless of Gleason grade, should be considered as clinically insignificant. However, in 15% of the cases, surgical margins were positive and extraprostatic growth was diagnosed in 9% of specimens. In addition to the definition of focal cancer used in these studies, further criteria for small cancer lesions have been proposed. Allan et al. defined focal cancer as 0.5 mm tumor in 1 biopsy core and no Gleason grade 4 or 5.23 Based on prostatectomy findings, Epstein et al. proposed even smaller volume, 0.2 ml as indication of clinically insignificant or minimal cancer, because some prostate cancers with the volume of 0.2–0.5 ml were not organ-confined.24
Postma et al.25 compared PSA progression after radical prostatectomy to PSA doubling time of watchful waiting policy among patients with focal cancer. The PSA doubling time is used as a variable to observe prostate cancer. Patients with focal cancer in biopsy have a small risk of PSA progression after radical surgery (4.6%). However, using a PSA doubling time <4 years, PSA progression was observed significantly more often in the watchful waiting group compared to PSA recurrence in the radical prostatectomy group (96% vs. 67%; p = 0.0005).
Bilateral cancers in biopsies may predict the tumor volume in the prostate. It has been shown to correlate with stage with bilateral cancers representing more advanced cases.22 In the current study, the cancers both in the first and the second round and the interval cancers were probably more often smaller in volume than those in the control group, as there were more focal and less bilateral cancers in these subgroups. In the control arm and in nonparticipant group, the cancers were more often bilateral and more aggressive as high-grade tumors with positive Ki-67 expression were more common. Bilateral cancers in every subgroup had higher Gleason score and expressed more Ki-67 antigen compared to those limited to a single lobe.
Gleason score correlates with tumor stage, and the prognostic value of Gleason grading system is substantial. Tumors with low Gleason scores2–4 have excellent prognosis, whereas the tumors with the highest scores8–10 have the worst prognosis. Gleason score 7 is often regarded as an intermediate category with a moderate progression risk. In screening latent cancers with lower Gleason scores are increasingly detected.20 These cancers might progress to more aggressive without curative treatment, although the clinical behavior still remains unclear and needs more research.
Gleason score was associated with Ki-67, which is in accordance with earlier findings of prostate cancer.10 Ki-67 is regarded as an independent prognostic value. Higher Ki-67 expression is associated with aggressive behavior in prostate and other cancers. In our study, as the bilateral cancers were the most proliferating cancers, they should be considered as the most aggressive cancer group. In the tumors among nonparticipants and the control arm showed higher proliferative activity and indications of large volume, which was probably the reason, why they were diagnosed beyond the screening.
Focal cancers generally expressed low levels of Ki-67. As we could analyze Ki-67 in only half of the focal cancers (56%), because of the limited amount of tumor tissue, the most aggressive tumors might be overrepresented in the sample. The smallest foci with the lowest Ki-67 might be excluded in this manner. This might explain why MIB alone has not been a very strong preoperative prediction marker in screen-detected cancers in general.26 However, our recent findings14 on radical prostatectomies suggest that Ki-67 can predict clinical behavior of tumors within identical Gleason score. In the present study, a small proportion of the focal cancers was highly proliferative. The cumulative incidence of focal cancers was over 4-fold higher in the screening arm compared to the control arm. It would be especially important to detect potentially aggressive cancers needing active treatment in this large group of patients. Further studies are needed to show, whether proliferative activity predicts PSA progression and final outcome in patients with watchful waiting. Ki-67 analysis might help to predict such cases of potential progression and to decide what would be the few cases of focal cancer in which an active treatment with curative intent could be a better option than a conservative management with watchful waiting or active surveillance.
Our results indicate that the biological aggressiveness of screen-detected prostate cancers is more often lower than in the control arm and nonparticipants. Further, cancers detected in the second screening round show fewer aggressive features than those from the first round. Further, few interval cancers have characteristics indicating aggressive behavior, which suggests that a 4-year interval may not be too long in prostate cancer screening. Measurement of proliferation might be used as an optional aid in the decision of treatment of screen detected low grade cancers.