Sarah Flury, Department of Urology, Box 800422, University of Virginia Health System, Charlottesville, Virginia, 22908, USA. e-mail: firstname.lastname@example.org
To determine if atypical small acinar proliferation (ASAP) represents minimally sampled prostate cancer not fully evaluated on a biopsy or a distinct pathological entity, by examining prostates removed at radical cystectomy, as a finding of ASAP of the prostate on needle-core biopsy is closely associated with the detection of cancer on subsequent biopsy.
PATIENTS AND METHODS
In all, 65 consecutive cystoprostatectomy specimens taken from June 1990 to March 2004 had prostatic material reviewed by one genitourinary pathologist (S.E.M.). The presence of high-grade prostatic intraepithelial neoplasia (HGPIN), ASAP, and adenocarcinoma was recorded. Foci of ASAP found in the absence of cancer were assessed with additional sectioning, high-molecular weight keratin (CK903), and α-methylacyl coenzyme A racemase (AMACR) immunohistochemistry.
In all, 24 of 65 specimens (37%) had adenocarcinoma. Of the 41 without cancer, 18 (44%) had neither HGPIN nor ASAP, 14 (34%) had HGPIN alone, three (7%) had ASAP alone (four foci), and six had both HGPIN and ASAP (15%). Two foci of ASAP were not present on any further sectioning. The remaining eight foci all lacked CK903 stain, indicating disruption of the basal cell layer. Of these eight, only five were present for the AMACR stain, all of which were positive. Two of these five developed into a lesion considered cancer on further sectioning.
ASAP identified in incidental prostates represented marginally sampled cancer in at least two of 10 foci assessed. The remainder could not be resolved as benign on further evaluation, and remained suspicious for malignancy.
The resurgent increase in the incidence of prostate cancer, with ≈ 232 090 new cases projected for 2005, is attributed to current screening practices and expanded needle-core biopsy techniques . At the same time, refined recognition of benign pathological mimics of cancer, such as atrophy, adenosis and high-grade prostatic intraepithelial neoplasia (HGPIN), complicate the diagnostic dilemma of small foci of atypical glands. In response to these challenges, attempts to define quantitative criteria and diagnostic features of minimal-volume adenocarcinoma on a biopsy were proposed [2–7], and a category for atypical but non-diagnostic foci was recognized .
Even at its inception, the diagnosis of atypical small acinar proliferation (ASAP) was highly predictive of a cancer diagnosis on subsequent biopsy , but its use recognises the histological overlap between small foci of neoplastic and non-neoplastic lesions. Immunohistochemical staining of basal cells using high-molecular weight cytokeratin (CK903) and/or p63, and for α-methylacyl coenzyme A racemase (AMACR) might confirm a histological impression, but neither stain provides a definitive diagnosis for prostate cancer.
Many recent publications have outlined the risk of prostate cancer in patients with HGPIN and/or ASAP in their prostate biopsies. Both are relatively rare diagnoses, with HGPIN found in 4–16% and ASAP in 1.5–2.5% of needle-core biopsies . HGPIN also suggests a higher risk of prostate cancer detection on subsequent biopsy, with a predictive value of 22–47%, but this is becoming increasingly controversial. Originally, studies reported that ASAP predicts approximately equally for prostate cancer and benign lesions on subsequent biopsies [5,9–13]; however, citing advances in immunohistochemistry and extensive sampling (increasing the diagnostic yield of a cancer diagnosis at first biopsy), a more recent report describes a decline to a predictive value of 37%.
By contrast, a series of immediate prostatectomies after an ASAP diagnosis found prostate cancer in all 25 cases (suggesting 100% predictive value), leading some researchers to advocate radical prostatectomy when ASAP is reported on core biopsy , hypothesising that ASAP is a biopsy artefact representing a shaved edge of cancer. Given the strong predictive value for subsequent cancer, probably exceeding that of HGPIN, there is speculation that ASAP represents a biopsy artefact of a marginally sampled prostate cancer focus. This is a distinct possibility, as this entity was created for, and has only been studied in, biopsy material, which because of its size and method of procurement is particularly vulnerable to this type of artefact.
We set out to test the hypothesis that ASAP merely represents under-diagnosed or ‘missed’ prostate cancer. If this is true, ASAP should only occur in biopsy specimens and never in prostatectomy specimens where full-tissue sections are available for analysis of the histological context. The corollary is that ASAP would never occur in a prostate gland that did not harbour prostate cancer. To evaluate this hypothesis, we reviewed prostates (removed at cystoprostatectomy) for foci of atypical glands comparable to what would be termed ASAP on a routine core biopsy, and evaluated the foci in a manner analogous to a diagnostic needle-core biopsy.
PATIENTS AND METHODS
A clinical record review identified 195 consecutive cystoprostatectomies carried out at a tertiary-care university hospital from June 1990 to March 2004. Of these, 65 (33%) patients had glass slides and formalin-fixed, paraffin wax-embedded archived blocks available for pathological re-review and analysis. These 65 patients comprised the study cohort. The study was approved by the University of Virginia Human Investigations Committee.
All 65 cases were examined by a genitourinary pathologist (S.E.M.) unaware of the original diagnosis. The slides were examined for the presence of cancer, HGPIN, and ASAP. Prostates with incidental prostate cancer were excluded from further review for HGPIN or ASAP. Foci of ASAP were further evaluated by immunohistochemical stains, and three additional levels cut from the corresponding formalin-fixed, paraffin wax-embedded archived blocks. For immunohistochemistry, tissue sections, including appropriate controls, were placed in citrate buffer (pH 6.0) and heated in a microwave oven for 20 min before separate application of antibodies to CK903 (clone 34βE12; 1 : 400 dilution; Dako, Glostrup, Denmark) and AMACR/P504S (1 : 200 dilution; BioCare Medical, Walnut Creek, CA, USA). After incubating with the primary antibodies, and adding the biotinylated secondary antibody, avidin- biotin immunoperoxidase was applied. Diaminobenzidine was used as the chromogen. Sections were then counterstained with haematoxylin. Strong nuclear immunoreactivity of CK903 was considered positive, while granular cytoplasmic immunoreactivity of AMACR was considered positive [16–21].
The clinical and demographic factors were analysed to assess whether the 65 patients later re-reviewed were representative of all 195 patients. Chi-square tests of association and the Wilcoxon rank-sum tests were used.
Using chi-square tests of association and Wilcoxon rank-sum tests, patient age (P = 0.92), presence of prostate pathology (P = 0.63) and presence of urothelial cancer in the specimen (P = 0.46) were not significantly different between the 65 re-reviewed patients and the 130 who were not re-reviewed. By contrast, procedure date was more recent for those re-reviewed (P = 0.012) and urothelial cancer stage (P = 0.003) was more likely to be T2-4 in the re-reviewed cohort (Table 1). The original diagnoses from the cystoprostatectomy and the diagnoses at re-review are outlined in Table 2.
Table 1. Comparison of the demographic and pathological features of all patients to those in the study cohort
The median (range) age of the 65 patients was 64 (42–86) years; the age of patients with incidental prostate cancer was 66 (45–84) years, with ASAP ± HGPIN was 64 (48–76) years, with HGPIN alone was 64 (53–86) years, and for the normal group (no cancer, ASAP or HGPIN) was 65 (42–82) years. The mean (range) date of surgery was 11 September 2000 (5 June 1990 to 19 March 2004). The patients had had cystoprostatectomy for TCC (35), squamous cell carcinoma (two), bladder cancer with no evidence of residual tumour at the time of cystoprostatectomy (26), colorectal carcinoma (one), and neuroendocrine tumour (one). The prostate pathology included Gleason 2–4 (three), Gleason 5 (five), Gleason 6 (11), Gleason 7 (four) adenocarcinomas.
On re-review, once prostate adenocarcinoma was identified, further pathological abnormality (HGPIN or ASAP) was not assessed. In all, 24 of 65 prostates (37%) contained prostatic adenocarcinoma. This is consistent with the published incidence of concomitant prostate and bladder cancer . Of the 41 benign prostates, three (7%) contained four foci of ASAP (Fig. 1), six (15%) contained both ASAP and HGPIN, and 14 (34%) contained HGPIN only. Figure 2 outlines the diagnoses. The 10 ASAP foci were further evaluated, with no information available for two of the 10 ASAP lesions, as they were not present on any additional level of sectioning and could not be further stained. The other eight ASAP foci were assessed with CK903 stain for a basal cell layer, all of which were negative. Of the eight foci of ASAP that showed absent CK903, three did not persist for the AMACR stain. All five of those that persisted were positive for granular cytoplasmic staining with AMACR. Of the five CK903 negative and AMACR positive lesions, two developed into histologically diagnosable prostate cancer with additional sectioning of the corresponding tissue block. These two foci had a total volume of much less than 0.5 mL, and had Gleason grades of 3 + 3. Figure 3 outlines the immunohistochemical analysis for the 10 ASAP foci.
The present study suggests that ASAP sometimes represents marginal sampling of prostate cancer missed on needle-core biopsy, because of small sample size and inability of the pathologist to analyse the sample in the full histological context. At other times, it reflects atypical glands that cannot be resolved as benign, but which are not unequivocally malignant even with all pathological material available for review. In many cases, atypical small proliferations can be resolved as benign with immunohistochemical staining of basal cells, using high-molecular weight keratin (CK903) and/or p63 [16,17]; however, patchy or absent staining can also be seen in benign mimics, such as adenosis and HGPIN, or can result from technical difficulties. The addition of AMACR, as a positive marker for prostate cancer, aids in evaluating small lesions, but can be negative in up to 18% of prostate cancers, and is generally positive in HGPIN [18–21]. Furthermore, lesions that disappear on subsequent levels, or stain equivocally, remain non-diagnostic.
A finding of ASAP on a needle-core biopsy alerts the urologist that the pathology is suggestive of cancer but that there is not enough data for a cancer diagnosis, and it properly triages patients into a high-risk category requiring very careful monitoring [10,23], as many are diagnosed with prostate cancer on subsequent biopsy [5,9–14]. Iczkowiski et al.  questioned whether prostate cancer diagnosed after an ASAP biopsy differed from prostate cancer diagnosed on initial biopsy. If all ASAP represents marginally sampled prostate cancer, these tumours might be expected to be smaller, and of lower Gleason score (predisposing them to be under-sampled or under-diagnosed). Their comparison of the subsequent prostatectomies found a difference only in the margin status of the two groups. In addition, the subsequent focus of prostate cancer should be found in the same location as the ASAP biopsy, but 39–53% are in a different sextant site [10,11].
A recent report on radical prostatectomy after a diagnosis of ASAP on biopsy found prostate cancer in all 25 prostates . However, while some of the prostate cancers were of advanced stage, others were <0.5 mL, which might indicate clinical insignificance . Those that were re-biopsied had a more typical follow-up profile, possibly indicating other contributing factors to explain the high risk of cancer, some of which were recently summarized by Zeliadt et al. . Together with our results, those data strongly suggest that, in most cases, ASAP represents marginally sampled prostate cancer. This model is entirely compatible with the findings of negative repeat biopsies in patients with ASAP, as the cancers are often of small volume .
In the present study, all eight ASAP lesions that persisted for at least one immunohistochemical stain had a profile consistent with prostate cancer, with absent CK903 staining and positive AMACR stains (Fig. 4), and two were confirmed to be marginally sampled prostate cancer by developing into a larger lesion on subsequent levels (Fig. 5). Assuming that an equivalent number of lesions would represent marginally sampled tumour with levels moving away from the lesion, causing it to disappear rather than expand, allows the extrapolation that 40% of atypical foci can be resolved as prostate cancer when the full histological context is available for review. In the present study, two of 10 ASAP samples were resolved as cancer and another six had an immunohistochemical profile consistent with cancer but could not, in our opinion, be quantitatively resolved as histologically diagnosable prostate cancer. It was also interesting that none of the ASAP foci developed into a recognisable benign lesion on subsequent levels. By evaluating full-tissue sections we could correctly categorize small atypical clusters in the histological context of atrophy, adenosis, radiation atypia, and budding HGPIN. In the absence of histological context, and with inadequate immunohistochemical evaluation, these can cause a diagnostic dilemma on core biopsy.
Some could argue that the six lesions with immunohistochemical support are all minimal prostate cancer. Because there is no clearly defined lower limit for diagnosis, this is a matter of experience and opinion. Findings from a recent questionnaire suggest that 61% of genitourinary pathologists have no lower limit for the diagnosis of prostate cancer, but even the authors speculate this would probably be in the context of clearly malignant architecture and cytology . This also refers to the interpretation of a diagnostic core biopsy, not a complete tissue section where the entire lesion can be visualized in two dimensions. Furthermore, to place these data in perspective, the eight foci that did not expand into prostate cancer, as well as the two foci that did, were all considerably <0.5 mL, proposed as a threshold for clinical significance . Given an interpretation of these lesions as an invasive cancer, they could be considered as potentially clinically insignificant.
The use and application of the diagnosis of ASAP created in the context of a diagnostic needle-core biopsy, as having atypical, but non-diagnostic glands, is of little clinical importance in an incidental prostatectomy specimen, and thus might be overlooked or disregarded in this context. By reviewing material with one genitourinary pathologist to find such lesions, we could adjust for the interobserver variability reported in HGPIN and ASAP diagnoses . Furthermore, we show that equivocal glandular proliferations are present in incidental prostates of men in the age range of those undergoing a diagnostic needle-core biopsy.
We would have liked to compare the incidence of these foci with those found in the prostates that did harbour cancer, speculating that a higher rate in the latter would support these lesions as something of a ‘field effect’ of prostate cancer, which is characteristically multifocal. We are in the process of examining the unaffected lobe of unilateral prostate cancer specimens for comparison with the current study.
In conclusion, although the clinical significance of ASAP on a diagnostic biopsy has been extensively researched and prognosticated, to our knowledge, no one has examined for the presence of small foci of atypical glands not associated with prostate cancer in whole-tissue sections. We found non-diagnostic foci in 22% (nine of 41) of incidental prostates, and with further analysis were able to prove that two of 10 ASAP foci represented marginally sampled prostate cancer, and six had immunohistochemistry supporting a cancer diagnosis, but not enough material for such a diagnosis. Although we have no way of determining the malignant potential of the remaining atypical foci, we show their presence in otherwise benign and asymptomatic prostates. We think our findings support an aggressive clinical approach to seek prostate cancer whenever ASAP is seen on needle-core biopsy, but the diagnosis reflects an equivocation and does not inherently imply clinically relevant under-sampled prostate cancer.