Stesura Seveso Archivio Italiano di Urologia e Andrologia 2014; 86, 4314 REVIEW Pathological issues in biopsy specimens of men with prostate cancer eligible for active surveillance Roberta Mazzucchelli 1, Andrea Benedetto Galosi 2, Antonio Lopez-Beltran 3, Marina Scarpelli 1, Liang Cheng 4, Rodolfo Montironi 1 1 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy; 2 Division of Urology, “Augusto Murri” General Hospital, ASUR Marche, Fermo, Italy; 3 Department of Surgery, Cordoba University Medical School, Cordoba, Spain; 4 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA. Active surveillance (AS) is an important management option for men with low- risk, clinically localized prostate cancer. The clinical parameters for patient selection and definition of pro- gression for AS protocols are evolving as data from sev- eral large cohorts become mature. Vital to this process is the critical role pathologic parameters play in identifying appropriate candidates for AS. These findings need to be reproducible and accurately reported by pathologists. Repeated biopsy after initial diagnosis of prostate cancer is recommended before inclusion in active surveillance for early detection of significant cancer. KEY WORDS: Prostate cancer; Prostate biopsy; Active surveillance; Tumour extent; Immunohistochemistry. Submitted 3 October 2014; Accepted 31 October 2014 Summary No conflict of interest declared. often with PSA kinetics and serial biopsy. Any progres- sion of the cancer while patients are monitored appears unlikely to threaten length of life (6). Through this report we aim at facilitating dissemination of information on the critical role pathological parame- ters play in identifying appropriate candidates for AS. CLINICAL PERSPECTIVE ON AS Specific inclusion criteria for AS vary across institutions (7, 8). Patients are selected for AS on the basis of their age, PSA density (PSA/prostate volume), measures of PSA kinetics, such as PSA velocity, percent of positive biopsy cores, the extent of prostate cancer in any core, and Gleason score 3 + 3 = 6 (9). Some of these cohorts include patients with intermediate-risk clinical parame- ters, allowing for inclusion of patients with PSA at diag- nosis greater than 10 ng/ml or including selected men with Gleason 3 + 4 = 7 PCa. Surveillance schedules for AS are variable across institutions. Most PCa experts agree that surveillance should include a combination of serial PSA and rectal examinations as well as repeat prostate biopsy. Biopsy grade reclassification has emerged as a more meaningful endpoint for men on AS. Higher-grade tumors (Gleason 7 and higher) clearly con- fer a higher likelihood of clinical progression. A finding of upgrading cancer on repeat biopsy commonly prompts treatment. This upgrading may represent undersampling at the time of initial diagnostic biopsy where the pre-existing high- grade prostate cancer was missed (10). Tables 1 and 2 include, as an example, inclusion criteria, surveillance schedule and the definition of progression of three AS protocols. REPEATED BIOPSY Repeated biopsy after initial diagnosis of prostate cancer is recommended before inclusion in active surveillance, since repeated biopsy improves cancer grading and reduce the risk of undersampling of significant cancer. Repeated biopsy should follow criteria of saturation DOI: 10.4081/aiua.2014.4.314 Presented at 19th National Congress SIEUN, Fermo 2014 INTRODUCTION The overwhelming majority of men diagnosed with prostate cancer (PCa) opt for primary curative therapy, such surgery to remove the prostate, i.e., radical prosta- tectomy, or radiation therapy to eradicate the tumor. However, most PCas are indolent and the number of newly diagnosed cases far outnumbering that of lethal cases (1). The magnitude of this so-called overdiagnosis, where cancers are identified that would never progress or cause harm to the patient if left untreated, ranges from 15% to 84% of new PCa cases (2, 3). Patients are exposed to the risk of overtreatment. Even if overtreat- ment is avoided, overdiagnosis induces anxiety associat- ed with the new cancer diagnosis, often resulting in fur- ther tests and expenses, and confers upon the patient a ‘cancer survivor’ label he carries for the rest of his life (4). Male offspring may worry about familial inheritance and increased risk of the disease. Given the indolent course of many PCa detected by PSA screening (5), active surveillance (AS) has emerged as an initial management alternative, thus men avoiding the side effects of PCa treatment. Under most AS strategies, patients undergo careful monitoring of the cancer, most Mazzucchelli_Stesura Seveso 15/01/15 11:01 Pagina 314 315Archivio Italiano di Urologia e Andrologia 2014; 86, 4 Pathology in prostate cancer active surveillance biopsy (20 core or more based on prostate volume) (11). Anterior gland should be included in the repeated biop- sy. Magnetic Resonance imaging can be performed before re-biopsy since the negative predictive value for significant cancer raise 95%, however the positive pre- dictive value is lower. Men who experience early upgrad- ing likely represent initial sampling error, whereas later upgrading may reflect tumor dedifferentiation. THE ROLE OF THE PATHOLOGIST The essential reporting items for cancer containing pro- static needle biopsies are listed in Table 3. Tumor extent measurements and the Gleason score are the most important pathologic parameters in needle biopsies determining eligibility for AS protocols. Tumor extent measurements There is no consensus on the best tumor quantification methods, which include: cancer percentage in each core, greatest percentage of cancer, cancer length in each core, greatest length of cancer (GLC), total percentage of carcinoma in all cores, total length carcinoma in all cores, fraction of positive cores, total carcinoma surface area and total percentage of carcinoma surface area in all cores. Tumor measurements are performed as a visual estimate or using an ocular micrometer or other morphometric measurement such as computerized methods. Visual estimation of percentage without morphometric meas- urements is commonly performed, although many recent studies do not actually describe whether visual estimation or morphometric measurements were used. Some use a regular ruler or the side graticule available on most microscopes for estimation of length and percent- age. The knowledge of the diameter of the field at each magnification for the microscope used to measure tumor extent can also help maximize accuracy of visual estima- tion of length. In a recent abstract, Mahamud et al. found no overall difference between visual estimation and measurement when determining percent involvement of prostate biopsies assessed only by whole slide images. However, there was a significant difference between the two methods when they considered a subset of cores deemed to have 40-60% involvement by visual estima- tion. It is unclear whether the accuracy of visual estima- tion of an image can be compared with that of a tissue core on a glass slide on a microscope. Data are conflict- ing whether morphometric measurements are superior to visual estimation and whether differences in the two methods would affect clinical management. Computerized morphometric measurements are consid- ered time-consuming and not practical for most pathol- ogists. Measurements of core length given in gross descriptions should not be used as these may not always be accurate. A few studies have assessed the value of the different methods of tumor extent measurement in prostate nee- dle biopsy in predicting pathological stage or prognosis. Quintal et al. (12) found that total percentage of carcino- ma in all cores and number and percentage of cores with cancer were significantly stronger than other methods Inclusion criteria Johns Hopkins University of Toronto PRIAS N° of patients 870 453 2494 Clinical stage T1c - ≤ T2 PSA density ≤ 0.15 ng/ml/cc - ≤ 0.20 ng/ml/cc PSA - ≤ 15 ng/ml ≤ 10 ng/ml No. of +ve cores ≤ 2 ≤ 2 % cancer per core ≤ %50 - Gleason score ≤ 6 ≤ 7 (3+4) ≤ 6 Surveillance schedule Institutions PSA and DRE Repeat prostate biopsy Definition of progression Johns Hopkins Every 6 months Yearly Gleason score > 6, or > 2 cores, or > 50% any core University of Toronto Every 3 months for 2 yrs, 6-12 months after diagnosis, PSADT < 3 years then every 6 months then every 3-4 years PRIAS Every 3 months for 2 yrs 1, 4, 7 and 10 yrs GS > 6, or ≥ 3 positive cores, and then every 6 months after diagnosis or PSADT < 3 yearly Table 1. Active surveillance inclusion criteria for selected institutions (see text). 1. Location of positive cores 2. Tumor extent 3. Gleason grades and score 4. Histologic type 5. Other (reported only if present) • Extraprostatic extension • Perineural invasion • Lymphovascular invasion • Intraductal carcinoma 6. Other atypical acinar foci suspicious for carcinoma Table 3. Essential reporting elements for cancer bearing prostatic needle biopsies. Table 2. Surveillance schedule and definition of progression for selected institutions (see text). Mazzucchelli_Stesura Seveso 15/01/15 11:01 Pagina 315 Archivio Italiano di Urologia e Andrologia 2014; 86, 4 R. Mazzucchelli, A.B. Galosi, A. Lopez-Beltran, M. Scarpelli, L. Cheng, R. Montironi 316 such as greatest percentage of cancer or length in a sin- gle core in predicting biochemical recurrence. Total per- centage of carcinoma in all cores had the strongest cor- relation and when combined with preoperative PSA and Gleason score improved prediction of pT3 in multivari- ate analysis. This was also independent for risk of bio- chemical recurrence. Bismar et al. (13) found that although many tumor measurements such as greatest percentage of cancer, total tumor length in millimeters, fraction of positive cores and total percentage of carcino- ma were significant in univariate analysis, only the frac- tion of positive cores was significant in multivariate analysis in predicting pT3 disease or positive margins. In this study all the measures were highly related to one another in a formal correlation analysis. Park et al. (14) examined the significance of the number of cores positive for cancer, percentage of positive biop- sy cores, total linear cancer length, total percentage of carcinoma and maximum cancer core length and found that, when considering PSA and Gleason score, none were significant in predicting pT3 disease in multivariate analysis. In a study by Brimo et al. (15), it was found that the fraction of positive cores, total percentage of carcino- ma and both total and greatest cancer core length were closely associated with pathological stage and biochemi- cal failure. The fraction of positive cores was found to be the factor most closely associated with pT3 disease in radical prostatectomy. Correlating needle biopsy cancer measurements with tumor volume in radical prostatectomy, Poulos et al. (16) found that the highest percentage of carcinoma in any biopsy site, percentage of adenocarcinoma at the biopsy site with the highest grading, the number of positive biopsy sites and tumor bilaterality were significant with the percentage of biopsy sites positive for disease the most significant predictor of tumor volume. In a study by Lewis et al. (17), tumor volume was best predicted by a combination of linear extent of carcinoma and number of positive cores. In a survey sent to 93 genitourinary pathologists the extent of cancer on needle biopsies was quantified by all the respondents with 80% reporting the number of cores involved by cancer. Linear extent was estimated by almost all, either as a percentage (80%) or millimeters of cancer length (41%) or both (22%). Considering the tumor quantification methods actually requested by urologists, in a 2005 study, 95% French and Belgian urologists requested the number of positive cores compared with 53% requesting length of cancer. In a study by Rubin et al. (18) 30 67% of urologists request- ed the percent involvement of each core by cancer, 33%, the number of cores with prostate cancer and 29% the length of core involvement. Recommendations by the College of American Pathologists, Association of Directors of Anatomic and Surgical Pathology and the World Health Organization for reporting carcino- ma extent have been summarized. Given these recom- mendations, the extent parameters currently in use in AS protocols and the evidence from the literature, it is sug- gested that pathologists should report the absolute num- ber of involved cores out of total number cores and the amount of cancer in the single core with the greatest amount of tumor expressed as the percentage involve- ment by carcinoma, with or without the linear extent of carcinoma in that core. Percentage involvement by carci- noma and or linear extent of carcinoma in each positive core may also be provided. All other measurements are optional. Linear extent of carcinoma in each core may also be provided. Other measurements are optional. The extent of cancer in prostate needle biopsy cores in patients potentially candidates for AS should based on the recording of: 1. Number of positive cores/total number of cores. The number of positive cores could possibly affect subse- quent therapy in terms of suitability for AS, such that it is justified to perform an immunohistochemical work-up of additional atypical foci (either HMWCK or p63 or combination of the two with AMACR). 2. Linear percentage of prostatic tissue involved and/or total linear measurement of carcinoma and total core length. This can be done calculating the percentage of each core involved by cancer, based on the linear length of cancer (mm) divided by the core length, then multiplied by 100. The other method is to pro- vide a percentage estimate of involvement of each of the cores derived by visual estimation. It has been shown that the former is more accurate and repro- ducible when patients are evaluated for eligibility for an AS protocol. PROBLEMS ASSOCIATED WITH TUMOR EXTENT MEASUREMENTS Measuring discontinuous foci of cancer When measuring discontinuous foci of cancer on a prostate needle biopsy core, the pathologist has to spec- ify presence of discontinuous foci, linear extent in aggre- gate of discontinuous foci, percentage involvement of the core, and the core length spanned by discontinuous foci. The following sentence is suggested as a template for the pathology report: “Prostate biopsy core (Length: 1.2 cm) with two discontinuous foci, measuring 1.5 and 1.2 mm (measuring 2.7 mm in aggregate), respectively, of Gleason score 3 + 3 = 6 acinar PCa separated by 4 mm of intervening benign tissue. The tumor spans 56% of the core length, involv- ing 22% of the core”. Tissue core and tumor fragmentation Concerning the number of cores per cassette, the ideal would one core per cassette. Two biopsies from the same location could be embedded together. It has been shown that simultaneous inclusion of 3 biopsies in the same cassette can lead to the loss of a mean length of 1.15 cm of assessable tissue which corresponds to the average length of one prostate biopsy. When multiple cores are submitted in a single cassette or jar by the urologist and processed in a single cassette, many pathologists give the overall percentage of cancer for the entire slide as opposed to the percentage for each individual core. At the Pathology Laboratory of United Hospitals, Ancona, we attempt to give the percentage of cancer per core for each individual positive core, regard- less of how many cores are on a given slide. Mazzucchelli_Stesura Seveso 15/01/15 11:01 Pagina 316 317Archivio Italiano di Urologia e Andrologia 2014; 86, 4 Pathology in prostate cancer active surveillance When reporting biopsies with multiple cores in the same jar, the pathologist has to provide linear measurement (in millimeters) or linear extent (as percentage) of pro- static tissue involved for the most involved core and the overall linear percentage or measurement in millimeters of tissue submitted from the site(s) with cancer. If there are multiple fragmented small cores containing cancer, an accurate assessment of percentage of cancer per core cannot be determined, and only an overall per- centage of cancer per fragmented specimen can be noted. In this scenario, one cannot even determine with cer- tainty the number of positive cores. There is evidence in the literature that there is a greater tendency to core frag- mentation when > 1 core is submitted in a container. It is our experience that needle biopsies collected onto gauze or paper are more likely to fragment. Assessment of number of cores involved by PCa is difficult in the presence of core fragmentation. A comment should be made suggesting that the urologist/clinician obtaining the biopsy is in the best position to make determination of number of cores involved based on the original sub- mission of number of cores, and, if necessary, to under- take clinico-pathologic correlation (19). Minimum acceptable core length Currently there is no definition for adequate or mini- mum acceptable core length. The percentage of cancer in a short core (e.g. < 5-10 mm) versus that in a sufficient- ly long core mean entirely different tumor lengths. This has implications for interpretation of percent core involvement in the setting of AS. Since percent core involvement is based only on total length of prostatic parenchyma, non-prostatic elements should not be included in total core length assessment. GLEASON SCORE The previous decade has seen considerable change in practice relating to Gleason grading of prostatic carcino- ma, and in 2005 the International Society of Urological Pathology (ISUP) undertook a major revision of the Gleason grading system. This was designed to reflect current practice and to incorporate recently gained knowledge on the biology of prostate cancer. The ISUP 2005 modification of the Gleason Grading System has resulted in changes to the definitions of Gleason patterns 3 and 4 tumors. This is of particular importance for those patients in which deferred treatment is contem- plated, as grade is central to the criteria utilized for iden- tifying patients suitable for inclusion in AS programs. Two features define Gleason pattern 3 glands: clearly infil- trating glands (in contrast to the overall nodular configu- ration of glands in Gleason patterns 1 and 2) and each gland being a single discrete individual glandular structure that is well formed. The 2005 ISUP Modification of the Gleason grading system defined virtually all cribriform glands as Gleason pattern 4, although in this classification well circumscribed, small, ovoid to round cribriform glands with regular bridging were included in Gleason pattern 3. More recently it has been suggested that as all cribriform glands appear to be associated with a less favor- able prognosis; these glands should also be classified as pattern 4. Glands with a glomeruloid architecture are also considered as pattern 4. Using these criteria, classification as Gleason pattern 3 should be confined to tumors con- sisting of well formed, separate glands with lumina. Accurate distinction of Gleason pattern 3 from Gleason pattern 4 is critical for eligibility for most AS protocols. When there is doubt, it is suggested: • Defaulting to a lower grade, • Following the focus on deeper serial sections, • Sharing such borderline cases with a colleague as the presence of a Gleason pattern 4 may preclude the patient from AS. Biopsy Gleason grade reclassification has emerged as one of the most meaningful interven- tion criteria for men on AS. SITUATIONS THAT SHOULD EXCLUDE A PATIENT FROM AS The presence of perineural invasion does not represent an exclusion criterion. However, there are rare patholog- ic situations that should likely exclude a patient from AS: • Histologic types: prostatic adenocarcinoma with pre- dominant ductal carcinoma histology, sarcomatoid carcinoma, small cell carcinoma • Intraductal carcinoma without invasive carcinoma • Extraprostatic extension in needle biopsy • Lymphovascular invasion in needle biopsy. CONCLUSIONS Given the overdiagnosis and overtreatment of low risk prostate cancer, AS should be a ubiquitously adopted and formalized strategy. 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Correspondence Roberta Mazzucchelli, MD r.mazzucchelli@univpm.it Marina Scarpelli, MD m.scarpelli@unvpm.it Rodolfo Montironi, MD (Corresponding Author) r.montironi@univpm.it Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, I−60126 Torrette, Ancona, Italy Andrea Benedetto Galosi, MD galosiab@yahoo.it Division of Urology, “Augusto Murri” General Hospital, ASUR Marche Fermo, Italy Antonio Lopez-Beltran, MD em1lobea@gmail.com Department of Surgery, Cordoba University Medical School Cordoba, Spain Liang Cheng, MD linag_cheng@yahoo.com Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Mazzucchelli_Stesura Seveso 15/01/15 11:01 Pagina 318