Section 7 -

Handling and Reporting Radical Prostatectomy Specimens Lars Egevad, MD PhD International Agency for Research on Cancer (IARC) Lyon, France

The purposes of the histopathological examination of radical prostatectomy specimens are to verify the preoperative diagnosis of cancer and to obtain information of prognostic importance, such as tumor type, grade, stage and margin status [1]. In academic institutions, there is also a need to sample tissue for research. Handling of these specimens was discussed by the pathology group at the WHO-sponsored International Consultation on Prediction of Patient Outcome in Prostate Cancer in Stockholm 2004 and the proceedings may serve as a guideline [2]. Recommendations have also been issued by The College of American Pathologists [3, 4]. Gross Examination and Handling of the Specimen General considerations There are a number of key issues that have to be addressed when deciding the local routine for handling prostatectomy specimens. Should the prostatectomy specimen be submitted to the laboratory in formalin or unfixed? For sampling of fresh tissue for research purposes, it is evidently necessary that the prostate is transported unfixed. In institutions with no such research activity, it might be more convenient to send the prostate to the pathology department in formalin. It is then important that the specimen is immersed in a large quantity of formalin (at least 500 ml). In these cases, fixation can be improved by injecting formalin into the prostate in the operation room (see below) [5]. Should the prostate be totally embedded? A problem when handling radical prostatectomy specimens is that cancer is often not visible at gross examination and the tumor extent is always underestimated by the naked eye. For the pathologist, the safest method to avoid under-sampling of cancer is to submit the entire prostate. However, this is time-consuming for the laboratory and in many institutions, partial sampling is practiced. This requires that the pathologist adheres to a strict protocol [3, 4, 6]. Should the prostate be wholemounted or should the slices be cut in smaller blocks to fit into standard cassettes? There are pros and cons with both methods. Disadvantages with wholemount sections include that recuts are more difficult to make and it is more expensive and difficult to perform immunohistochemistry. Tissue microarrays can be constructed from wholemounts for immunohistochemistry but this technique damages the paraffin blocks and it is a rather time-consuming process to set up a tissue microarray experiment on prostate cancer. Moreover, wholemount sections do not fit into standard slide holders for slide collections and standard slide archives. Yet, wholemount sections give the pathologist a better overview and the identification of multiple separate tumor foci is facilitated. Lab technicians who are used to cut wholemounts usually find them less time-consuming than cutting multiple small blocks. Handling of unfixed prostatectomy specimens The fluid secreted by the prostate gland is known to contain abundant proteolytic enzymes causing rapid proteolytic degradation. Hence, the prostate should be immersed in ice immediately after surgery and rapidly transported to the pathology department. Formalin fixation should be performed as soon as possible after harvesting of research samples (see below). Sampling for research purposes Several techniques have been described for harvesting of fresh tissue. Samples can be punched by core needle or punch biopsies [5, 7, 8] but the tissue yield with this method is small. It is also difficult to localize cancer when biopsies are taken from the outside of the prostate gland. If the prostate is incised (e.g. cut in half by a horizontal section), the cut surfaces can be inspected and sampled where tumor is seen grossly [9]. To prevent deformation of the prostate during fixation, the prostatic capsule is pinned out and wholemount sections can still be obtained. If the unfixed prostate is cut into multiple small blocks for submission in standard cassettes, sampling can easily be done from any grossly visible cancer. Formalin fixation Formalin injection into the specimen before immersion in formalin improves fixation [5]. In a study from our group (unpublished data), there was no difference in tissue shrinkage between specimens injected with 20 ml formalin and prostates that underwent conventional fixation. Hence, the same volume correction factor may be used for planimetrical tumor volume measurements. No needle tracks or other traces of the injection (such as edema) are seen in the histological sections. The prostate is then immersed in formalin in a large jar. Fixation can be enhanced by circulating the formalin with an aquarium pump. After fixation the prostate should be inked. There are several commercially available ink systems. It is recommended that several different colors (at least 3) are used in order to facilitate orientation. The adhesion of the ink can be improved by dipping the prostate in 5% acetic acid. The apex The apical margin is a common location for positive margins and should be examined carefully. The shave technique is no longer recommended [2]. A better method is to amputate a thick section from the apex and then cut it sagittally. The base Positive surgical margins are less often seen in the base area than at the apex. Therefore, the base may either be examined by sagittal sections as described for the apex, or a thin shave slice may be removed and embedded en face. [2] Slicing of the prostate After the apical slice has been removed, the prostate is sliced at 3 - 4 mm. An electric meat cutter is helpful when slicing the formalin fixed prostate. However, there is a risk that the cutter removes the surgical margin at the end of the slice. This can be avoided if the last part of each slice is cut by hand. The seminal vesicles The seminal vesicles and vasa deferentia may be cut longitudinally and both halves embedded. However, seminal vesicle invasion is nowadays relatively rarely seen and complete embedment of these structures is not mandatory. As seminal vesicles are usually invaded through their attachment to the prostate, a minimum requirement is that a section through the base of the seminal vesicle is submitted. Gross features of Prostate Cancer Prostate cancer is often difficult to see grossly and this is particularly true for small tumors. When cancer can be identified at gross examination, it is typically very poorly circumscribed. The color of the tumors ranges from bright yellow to pale tan. Sometimes the tumor is merely firmer than normal prostatic tissue but has normal color. Transition zone cancers are especially difficult to see [6]. They typically grow within hyperplastic nodules and the nodularity of pre-existing hyperplasia is preserved. The only sign of cancer may be that a transition zone nodule is slightly more yellow than normal. The microscopic extension of prostate cancer is almost always greater than expected from the gross features. Reporting of Radical Prostatectomy Specimens Extra-prostatic extension The prostatic capsule is a poorly defined structure and not a true capsule. The prostatic "capsule" refers to the transition between relatively condensed prostatic stroma and more loosely arranged extra-prostatic connective tissue. Hence, extra-prostatic extension is a better terminology than capsule penetration. Extra-prostatic extension is most common in the dorsolateral region at the neurovascular bundle. In most cases of extra-prostatic extension, there is tumor growth in adipose tissue. However, as opposed to core biopsies, prostatectomy specimens may be diagnosed with extra-prostatic extension even when extra-prostatic cancer is surrounded by desmoplastic connective tissue. The capsule is particularly poorly defined around the apex, anteriorly and at the bladder neck, and in these areas, growth in or at the level of adipose tissue is required for a diagnosis of extra-prostatic extension. At the apex and anteriorly, there is a continuous transition between prostatic tissue and striated muscle tissue of the pelvic floor. Hence, growth around striated muscle does not necessarily indicate extra-prostatic extension. Extra-prostatic extension can be stratified as focal vs. established (or extensive or non-focal) [10, 11]. Patients with focal extra-prostatic extension have a more favorable outcome after radical prostatectomy. However, there is no uniform definition of these categories: measures such as "a few glands outside the prostate" [11] or "less than one high-power field" [10] have been used to define focal extra-prostatic extension. Surgical margins The prostatectomy specimen is only surrounded by a thin layer of connective tissue and a wide surgical margin cannot be expected. For a margin to be considered positive, cancer has to reach ink. A margin is reported as negative if cancer is separated from ink by as little as a few collagen fibres. Positive margins are most common at the apex but may occur anywhere. Seminal vesicle invasion Cancer invading the seminal vesicle conveys a poor prognosis. Cancer usually invades by direct overgrowth from the basal parts of the prostate. Three anatomical patterns of seminal vesicle invasion have been described [12] but the prognostic value of these categories has not been confirmed by others [13]. Zonal origin and distribution It has been suggested that transition zone (TZ) tumors have a better prognosis than peripheral zone (PZ) tumors of the same grade and size, possibly because of the greater distance to the capsule. Hence, the zonal origin of prostate cancer should be reported in prostatectomy specimens. When the tumor is large and extends into both the PZ and the TZ, it may be impossible to determine its origin. Tumor volume There are several methods for estimation of tumor volume, including planimetry, the grid method, the percentage of the specimen involved by cancer and maximum tumor diameter. Volume of prostate cancer correlates with other prognostic factors such as grade, stage and ploidy and also with prognosis after radical prostatectomy. Some authors have reported that tumor volume is not an independent predictor of prognosis when Gleason score, extra-prostatic extension, surgical margins and seminal vesicle invasion are included in the analysis [14, 15]. This may be true when the majority of tumors are small. Other studies based on series with larger tumors have found that tumor volume is an independent prognostic factor [16]. Because of these conflicting data, measurement and reporting of tumor volume is not mandatory. Grading of Prostate Cancer in Radical Prostatectomy Specimens The histopathological tumor grade is the most important tissue-based prognostic factor of prostate cancer. The Gleason grading system is now uniformly used for grading of prostate cancer and has been adopted by the WHO [17]. Detailed descriptions of this grading are given on several web resources such as: http://pathology2.jhu.edu/gleason/ http://www.pathology.ks.se/egevad/gleason.html The Gleason patterns Gleason scores 2 and 3 are only exceptionally assigned, because Gleason pattern 1 is unusual. Gleason score 4 is also relatively uncommon because pattern 2 is usually mixed with some pattern 3 resulting in a Gleason score 5. Normal prostate epithelial cells are arranged around a lumen. In patterns 1 to 3, there is retained epithelial polarity with luminal differentiation in virtually all glands. In pattern 4, there is partial loss of normal polarity and in pattern 5, there is an almost total loss of polarity with only occasional luminal differentiation. The 2005 ISUP Consensus Conference on Gleason Grading of Prostatic Carcinoma At an ISUP (International Society of Urological Pathology) consensus conference in 2005, the Gleason grading system for prostatic carcinoma underwent its first major revision including both pattern interpretation and compiling and reporting of grade information [18]. The most important modifications of pattern interpretation proposed by the ISUP meeting pertained to patterns 3 and 4. In the original description by Gleason, cribriform patterns were assigned either a Gleason pattern 3 or 4 depending on the shape of the cribriform glands. There is now an increased understanding that invasive cribriform carcinoma is a relatively aggressive disease. Therefore, it was now recommended that most cribriform carcinomas should be assigned a Gleason pattern 4 rather than 3 and that cribriform glands must be small and round to qualify for pattern 3. The ISUP consensus meeting emphasized that Gleason pattern 4 should also include ill-defined glands with poorly formed glandular lumina, a relatively common pattern that was not incorporated in the original definition of pattern 4. Reporting of tertiary Gleason patterns Gleason defined the Gleason score as the sum of primary (dominant) and secondary (next most common) patterns. However, a recommendation was issued at the ISUP meeting that Gleason scores of needle biopsies should include any amount of high-grade tumor, even if less than 5% of all cancer present [18]. This may be justified because grading of needle biopsies serves as a guideline for the clinician on how to handle the patient rather than providing a definitive Gleason score. When reporting a radical prostatectomy specimen, the enitre cancer is available for grading and there was a consensus that for radical prostatectomy specimen, one assigns a Gleason score based on the primary and secondary patterns with a comment when a tertiary pattern is present. Grading of separate tumor foci Prostate cancer is a strikingly multifocal disease. The recommendation of the ISUP consensus meeting was that separate Gleason scores should be assigned when more than one major tumor focus is present. If for example the dominant tumor focus is a Gleason score 4+4=8 peripheral zone cancer and there is a separate Gleason score 2+2=4 transition zone cancer, the prognosis of the high-grade cancer is not improved by the presence of the low-grade tumor. Hence, it would be misleading to assign a Gleason score 4+2=6. Crush artefacts Crush artefacts are particularly common at the margins of radical prostatectomy specimens. Crush artefacts cause disruption of the glandular units and consequently may lead to overgrading of prostate cancer. These artefacts are recognized by the presence of noncohesive epithelial cells with fragmented cytoplasm and dark, pyknotic nuclei adjacent to preserved cells. Crushed areas should not be Gleason graded. Gleason score and prognosis Multiple studies have confirmed that Gleason score is a very powerful prognostic factor, both for prediction of the natural history of prostate cancer [19, 20] and for assessment of the risk of recurrence after radical prostatectomy [21, 22] or radiotherapy [23]. The major increase of the likelihood of having adverse findings in the prostatectomy specimen or with failure following prostatectomy or radiotherapy is from Gleason score 6 to 7. 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