Introduction 9 vanishing cancer phenomenon 5 7 30 5 4 4 28 32 25 13 13 6 11 12 15 16 19 24 26 11 16 28 The aim of this paper was to report the results of a search for residual cancer on RP after an initial stage pT0 evaluation in eight patients with positive biopsy. Materials and methods We reviewed our database of 1,328 consecutive patients whose biopsies and RP specimen were examined between March 1995 and June 2006 at the five Pathological Anatomy Services associated with the Polytechnic University-United Hospitals of the Marche Region, Ancona, Italy. Most of the biopsies and all the RPs of this series of 1,328 patients were reported by the same pathologist (RM). Seven hundred patients were untreated before the operation, whereas 628 had received neoadjuvant endocrine treatment for approximately 3 to 6 months. 28 17 18 28 We identified eight patients with positive 6- to 12-core biopsy and with no residual cancer (pathological stage pT0) in the initial routine histological examination of the RP and successively subjected to additional sectioning and evaluation. Search for residual cancer 18 The diagnostic needle biopsies were reviewed to exclude the possibility of a false positive biopsy diagnosis and to assess the approximate location of the biopsy with tumour, such as apex, mid-zone and base, both left and right. The slides of the surgical specimens were reviewed for residual cancer that was initially overlooked or missed. If the prostate was not totally embedded, the remaining prostate tissue was processed in toto. If the prostate was completely sampled, then this step was skipped. This type of information was usually contained in the pathology form where the all the steps of the processing procedure were recorded. The information was further confirmed by searching the specimen’s container for residual pieces. As we routinely sample completely all prostatectomies, this step was skipped. Additional deeper sections (i.e. three to five sections) of the prostatectomy area (paraffin block) corresponding to the location of the core with cancer were re-cut. Further sections were also obtained from all the other paraffin blocks. Additional deeper sections (i.e. three to five sections) of the area corresponding to the location of the positive core as well as of all the remaining blocks were re-cut after block-flipping. Immunostains for p63 and alpha-methylacyl-CoA racemase (AMACR) were performed to evaluate suspicious foci (When these two were not yet available, we used 34betaE12 immunostaining). 17 Review the description of the macroscopic appearance of external and cut surfaces of the surgical specimen as well as inspect the contour of the tissue sections on the slides for hint or clues that might indicate that part of the tissue was missing either due to the surgical procedure or for technical reasons. DNA specimen analysis was performed on formalin-fixed tissue to confirm the identity of the biopsies and prostatectomies whenever necessary. DNA specimen identity analysis Tested samples in an individual case included the biopsy core with cancer and a random block for the corresponding RP. The tissue was obtained as a direct section from formalin-fixed and paraffin-embedded blocks. DNA was extracted from paraffin-embedded tissue using the QIAmp DNA mini kit (Qiagen) according to manufacturer’s protocol (Promega, Madison, WI, USA). Fifteen microsatellites and amelogenin locus were co-amplified by the AmpFlSTR identifiler kit (Applied Biosystems, Foster City, CA), and the amplified fragments were electrophorized on an ABIPrism3130 genetic analyzer (Applied Biosystems). Fragment sizing and allele designation were established by GeneMapperID v3.2 software (Applied Biosystems), and genetic profiles from both biopsy and prostatectomy samples were compared. Results Residual cancer was not found in eight RPs after an initial routine examination. They represent 0.6% of the 1,328 consecutive patients. Three (0.2%) of them were from the group of untreated patients, whereas five (0.4%) were from those who had received neoadjuvant treatment. The latter figure represents 0.8% of the treated patients. 1 Table 1 Patients’ clinical data Patient no. Age (years) PSA Prebiopsy (ng/ml) Digital rectal examination TRUS Gland volume (cc) 1 68 9.0 Abnormal Normal 47.9 2 71 5.4 Normal Abnormal 31.4 3 64 6.1 Normal Normal 28.1 4 67 1.25 Abnormal Normal 93.5 5 60 5.9 Normal Normal 36.6 6 66 3.0 Abnormal Normal 47.9 7 58 3.9 Normal Abnormal 29.3 8 66 6.5 Normal Normal 36.6 Mean (range) 65 (58–71) 5.1 (1.25–9.0)   43.9 (28.1–93.5) PSA TRUS 2 1 Table 2 Biopsy findings Patient no. No. of positive cores Gleason score Cancer length (% of involvement) Positive core location 1 1/12 3 + 3 = 6 5 Right apex 2 1/10 3 + 4 = 7 10 Right apex 3 1/6 3 + 3 = 6 5 Left mid-zone 4 1/6 3 + 3 = 6 5 Right mid-zone 5 1/6 3 + 3 = 6 5 Right mid-zone 6 1/12 3 + 3 = 6 10 Left mid-zone 7 1/10 3 + 3 = 6 5 Right mid-zone 8 1/12 3 + 3 = 6 5 Left base Fig. 1 a b 2  Search for residual cancer There were no cases with a false positive biopsy diagnosis (step 1). Cancer was not overlooked or missed in any of the eight prostatectomies (step 2). Each prostate had been totally embedded (step 3). 2 Fig. 2 a b c d e a b dotted area red arrow 1 b c d e  3 3 Fig. 3 scattered  4 3 4 Fig. 4 Whole mount section (case no 7). Part of the peripheral zone, posteriorly, is missing  Table 3 Results of the search for residual cancer Patient no. Neoadjuvant treatment Cancer found ID test done 1 No In recut No 2 No In recut No 3 Yes In recut No 4 Yes In recut No 5 Yes In recut after block-flipping No 6 Yes After cytokeratin stain No 7 Yes a Yes, identical 8 No No Yes, identical a Discussion 16 28 11 13 16 Additional sectioning and evaluation of the cases can reduce the number of pT0 RPs after a positive biopsy. In particular, the current study showed that the final incidence was 0.15% and included only two RPs with missing parts, probably due to incomplete removal of the prostate. One of these two patients was hormonally treated preoperatively. 8 2 22 21 1 10 23 28 31 28 29 28 28 6 14 20 27 There are four aspects that we have not explored in our study. One is that the carcinoma could be lost in facing off of the paraffin blocks. We are fully aware of this potential problem. Our technicians are instructed so that they have to collect tissue sections and not waste material when the paraffin blocks are levelled off. The second is whether the time, effort and expense of the sampling described in this paper are warranted on a routine basis for all pT0 cases. We have not done any analysis in these respects due to the fact that the number of cases is very small and that time and expense do not represent an issue of concern in our institution. The third is what degree of sampling would be necessary to serially section through the entire prostate. Probably thousands of sections would be required, and we were prepared to cut as many sections as needed to find cancer. The fourth is whether there is an outcome difference when the initial pT0 carcinomas are detected after more thorough sampling vs pT0 cases without additional sampling. This was not addressed in our study because the basic aim was to avoid that a pT0 report is rendered to the clinician and to the patient, thus triggering a potential legal issue with all the problems related to it. 9 3 Conclusions and recommendations The current study showed that an extensive search for residual cancer reduces the number of pT0 RPs after a positive biopsy. To achieve this, it is recommended to have the needle biopsy reviewed, carefully look again at the radical prostatectomy, do deeper sections and then flip certain paraffin blocks. In addition, atypical foci should be stained for basal cell markers and often AMACR, especially in hormone-treated cases. If a block is missing part of the peripheral zone (capsular incision), this should be commented on. DNA analysis for tissue identity should be performed when the other steps have been taken without finding cancer.