International Archives of Urology and Complications
A Comparison of Prostatic Acinar Adenocarcinoma Gleason 3+4 versus 4+3 in Different Laboratory Practice Settings
Celia Maria Jesuino Bittencourt1-3, Daniel Abensur Athanazio4-6*, Andréia Carvalho dos Santos4,5, Érica Curvelo Dantas Senna4 , Tânia Maria Correia Silva5 and Luiz Antonio Rodrigues de Freitas3-6
1Centro Estadual de Oncologia (CICAN), Av Vasco da Gama s/n, Brazil
2CLINAZZA Pathology Laboratory, Rua do Cabral, Brazil
3IMAGEPAT Pathology Laboratory, Rua Lucaia, Brazil
4Department of Pathology, Largo do Terreiro de Jesus s/n, Federal University of Bahia, Brazil
5Gonçalo Moniz Research Center, Rua Waldemar Falcão, Brazil
6Hospital Universitário Professor Edgard Santos (HUPES), Rua Augusto Viana s/n, Brazil
*Corresponding author: Daniel Abensur Athanazio, Departamento de Patologia e Medicina Legal, Universidade Federal da Bahia, Faculdade de Medicina da Bahia, Praça XV de novembro, s/n - Largo do Terreiro de Jesus, 40025010 - Salvador, BA, Brazil, Tel: 55 (71) 32835563, Fax: 55(71) 32408406, E-mail: email@example.com
Int Arch Urol Complic, IAUC-1-003, (Volume 1, Issue 1), Research Article; ISSN: 2469-5742
Received: February 05, 2015 | Accepted: March 18, 2015 | Published: March 20, 2015
Citation: Bittencourt CMJ, Athanazio DA, Santos ACD, Érica Curvelo Dantas Senna ECD, Silva TMC, et al. (2015) A Comparison of Prostatic Acinar Adenocarcinoma Gleason 3+4 versus 4+3 in Different Laboratory Practice Settings. Int Arch Urol Complic 1:003. 10.23937/2469-5742/1510003
Copyright: © 2015 Bittencourt CMJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Objective: To evaluate Gleason 7 tumors from radical prostatectomy specimens that were stratified by 3+4 and 4+3 status, focusing on the clinical and morphologic features.
Methods and results: In the first series of 20 Gleason 7 tumors processed by total embedding, cases with 4+3 status were associated with a higher proliferation index. In a second series of 106 Gleason 7 tumors processed by partial embedding, 4+3 status was associated with higher preoperative serum prostate-specific antigen levels and a higher frequency of extraprostatic extension and seminal vesicle invasion.
Conclusion: For those laboratories that employ partial samplings as the adopted method for processing radical prostatectomy specimens, inclusion of the entire prostatic periphery and extraprostatic tissue is advised to avoid understaging caused by missing extraprostatic extension.
Prostatic Neoplasms, Pathology, Prostate-specific antigen, Neoplasm Grading
The Gleason grading system is the method used to grade acinar adenocarcinoma of the prostate . Although a Gleason score of 7 is one of the most frequent scores reported in radical prostatectomy specimens, tumors with Gleason score 7 show heterogeneous behavior on follow up. Approximately 14 years ago, a body of literature began to accrue that explored the importance of distinguishing a final Gleason score of 7 based on Gleason 3 pattern predominance (3+4) or Gleason 4 pattern predominance (4+3). Several studies have shown varying associations with respect to morphologic parameters and prognosis when comparing Gleason 3+4 and 4+3 status. Some of these differences might be attributed to specific methods of radical prostatectomy specimen handling and evaluation. The present study compared the clinical and morphologic features of Gleason 7 tumors from radical prostatectomy specimens stratified according to 3+4 or 4+3 status. Specimens were obtained from two different series using distinct protocols: one employed total embedding and an original point-count method to assess tumor burden , and a second series used partial embedding and an adapted point-count method .
Material and Methods
Patients and protocols
The study included two series of consecutive radical prostatectomy specimens evaluated in Salvador, Brazil. The first series comprised 48 specimens collected from January 2000 to December 2003: 34 from Hospital Universitário Professor Edgard Santos (HUPES), 12 from CLINAZZA Pathology Laboratory and two from Centro Estadual de Oncologia (CICAN). All of these prostatectomy specimens were processed by complete embedding. The cone method was used to assess each specimen’s apical and basal margins, while the original point-count method described by Billis and colleagues was employed to evaluate tumor extent and the percentage of prostate involvement by carcinoma .
The second series comprised 224 radical prostatectomy specimens from the IMAGEPAT Pathology Laboratory (Salvador, Brazil), obtained from May 2010 to May 2013 (n=195), and 29 specimens from HUPES (2013). All specimens were processed by partial embedding with the cone method to assess the apical and basal margins and an evaluation of the tumor extent (and the percentage of prostate involvement by carcinoma) was performed using a recently described modified point-count method . This project was approved by the Research Ethics Committee of Centro de Pesquisas Gonçalo Moniz (CPqGM/FIOCRUZ) in Salvador, Brazil.
Paraffin-embedded sections were prepared at 4-µm thickness. Paraffin was removed by xylene, followed by rehydration with graded ethanol. Heat-induced antigen retrieval was carried out for all sections in 0.01?M citrate buffer, pH=6.00, at 95°C for 30?min. After washing in PBS with 0.1% Tween-20, subsequent steps were performed with the LSAB (DAKO) kit. The sections were incubated with the primary antibodies overnight at 4°C in a humidified chamber. Primary antibodies (mouse IgG, clone MIB-1; DAKO, Carpinteria, USA) were diluted in antibody diluent Af (2% BSA in PBS; pH 7.4; DAKO). After washing in PBS, the sections were incubated in 10% skim milk for 20 min to block non-specific binding. A blockade of endogenous peroxidase was achieved with 3% H2O2 for 10 min at room temperature. Reactions were developed with 3,3-diamino-benzidine tetrahydrochloride (DAB) (DAKO). Sections were counterstained with Harris hematoxylin for 2 min, then dehydrated and mounted using Permount.
Statistical analyses were performed using the GraphPad Prism 4.03 software package (GraphPad,La Jolla, CA, USA). Categorical data were compared by Chi-square test or Fisher´s exact test, and the numerical data were compared using the Student´s t-test or the Mann-Whitney test. The Chi-square test for trend was used to compare stratified groups with different serum Prostate-specific Antigen (PSA) levels and the percentage of gland involvement by carcinoma.
In the first series, 20 of 48 (42%) radical prostatectomy specimens exhibited tumors with Gleason score 7. In this series, we observed a trend (p=0.06) with respect to higher serum preoperative PSA levels in patients with 4+3 status. Five of seven (71%) patients with 3+4 status had serum PSA levels between 4.00 and 9.99 ng/ml, whereas 5/7 (71%) patients with 4+3 status had serum PSA levels > 9.99 ng/ml. Significant differences were observed between the groups regarding the percentage of gland involvement by carcinoma (PGIC) and the percentage of cells stained for a cellular marker of proliferation (Ki-67 antigen immunostaining). Seven of nine patients with 4+3 status (77%) had PGIC > 20%, whereas 7/11 patients with 3+4 status (64%) had PGIC ≤ 20% (p=0.03). Two of nine patients with 4+3 status (22%) had > 3.0% cells stained positively for Ki67, whereas 11/11 patients with 3+4 status (100%) had ≤ 3.0% cells stained for Ki67 (p=0.04). There were no differences observed between the groups regarding circumferential margins, basal margin, apical margin, extraprostatic extension or seminal vesicle invasion. No tertiary Gleason 5 component was observed in this series. Data from the first series are detailed in Table 1. Among these 48 prostatectomy specimens, the percentage of Ki67-positive cells was positively correlated with PGIC and Gleason grade.
Table 1: Clinical and morphologic features of prostate carcinoma with prognostic value in cases of prostatic acinar adenocarcinoma Gleason 7 stratified by 3+4 or 4+3 sum. Specimens were processed by complete embedding and evaluated by the original point-count method (n=48). View Table 1
We extended the comparison of 3+4 and 4+3 status in a larger series of radical prostatectomies processed by a partial sampling method and using an adapted modified point-count method to estimate PGIC and tumor volume. In this series, 106 of 224 (47%) radical prostatectomy specimens contained tumors with Gleason score 7. Patients with 4+3 status had higher preoperative serum PSA levels. Despite a lack of differences in PGIC and tumor volume, tumors with 4+3 status were more likely to exhibit extraprostatic extension and seminal vesicle invasion. The rate of tertiary Gleason 5 component detection was similar in each group. Data from the second series are detailed in Table 2.
Table 2: Clinical and morphologic features of prostate carcinoma with prognostic value in cases of prostatic acinar adenocarcinoma Gleason 7 stratified by 3+4 or 4+3 sum. Specimens were processed by partial embedding and evaluated by the adapted point-count method (n=106). View Table 2
In 2000, in a series of 567 radical prostatectomies for localized acinar Gleason score 7 adenocarcinoma, Partin and colleagues reported that Gleason 4 pattern predominance was associated with lower recurrence-free survival intervals. The tumor 4+3 status was an independent predictor of serum PSA progression . That same year, Sakr and colleagues reported a series of 534 patients with Gleason 7 tumors. Patients with 4+3 status were more likely to be older African-Americans and exhibited higher preoperative serum PSA levels, a higher stage and larger tumors. In a multivariate analysis among patients with organ-confined disease, primary Gleason 4 status was the only independent predictor of PSA recurrence . In 2001, in a series of 237 Gleason score 7 tumors, Lau and colleagues reported that 4+3 status was associated with seminal vesicle involvement, a higher stage, extraprostatic extension and higher preoperative serum PSA levels, but not with cancer-specific survival. Under multivariate analysis, preoperative PSA, seminal vesicle involvement and DNA ploidy – but not the primary Gleason pattern – were associated with progression-free survival . In 2001, Herman and colleagues reported a series of 823 patients with Gleason 7 tumors treated by radical prostatectomy in which primary pattern 4 was associated with lower recurrence-free survival. However, multivariate analysis, including variables such as preoperative PSA, tumor volume, margin status, seminal vesicle involvement, extraprostatic extension and nodal metastasis, did not show that Gleason 4 pattern predominance had the predictive power to be independently associated with disease progression .
More recently, some authors have called attention to the importance of tertiary pattern 5 in radical prostatectomy specimens. In a series of 228 patients with Gleason score 7, 4+3 status was associated with a higher stage, serum PSA recurrence and the presence of tertiary Gleason 5 patterns. Using multivariate analysis, tertiary pattern 5 (but not primary Gleason pattern) was an independent predictor of PSA recurrence . In another series of 509 patients with Gleason score 7, tertiary pattern 5 was independently associated with a higher stage and PSA recurrence. The impact of tertiary pattern 5 on PSA recurrence was observed in both 4+3 and 3+4 subsets . Interestingly, in the present study, the frequency of tertiary Gleason 5 did not differ between 3+4 and 4+3 status.
The long-term prognostic value of stratifying Gleason 7 tumors was evaluated in 1,256 men in a report from the Mayo Clinic. After ten years, patients with 4+3 and 3+4 status differed in terms of PSA recurrence-free survival (38% vs 48%), systemic recurrence (15% vs 8%) and cancer-specific survival (93% vs 97%). Primary Gleason patterns among patients with Gleason score 7 remained an independent predictor of all these endpoints when controlling for preoperative PSA, seminal vesicle involvement, margin status, DNA ploidy and TNM staging . In a more recent series of 756 men with prostate cancer, cancer-specific survival after ten years was 98%, 92%, 77% and 70% for patients with tumors of Gleason score ≤ 6, 3+4, 4+3 and ≥ 8, respectively. Another study found that, in patients with Gleason score 7, the primary Gleason pattern was an independent predictor of cancer-specific survival .
In a series of 530 patients who underwent brachytherapy for Gleason 7 tumors, the primary Gleason pattern was not found to be predictive of PSA recurrence or survival . It is worth noting, however, that primary Gleason patterns assessed in needle biopsy specimens exhibit a well-recognized level of discrepancy when compared to the (usually upgraded) Gleason score obtained from radical prostatectomy specimens. Thus, at least in part, sampling error may contribute to the lack of an effect observed in patients treated by brachytherapy.
Discrepancies reported in the literature, in which factors emerge as independent predictors of outcome, are dependent on which particular variables were included in a given study. Additionally, certain differences between studies might be attributed to different methods of radical prostatectomy specimen handling and examination. In this study, we demonstrated that Gleason grade 7 tumors with 3+4 and 4+3 status exhibited important differences, even when evaluated by different protocols.
We highlight the observation from the first series that Gleason 7 tumors with 4+3 status were associated with higher proliferative indices. Importantly, Ki67 expression has been reported as the most consistent protein/immunohistochemical marker associated with prognosis in prostate cancer [13,14]. From the second series, which was powered by a larger sample, 4+3 status was associated with extraprostatic extension and seminal vesicle invasion.
This study has several limitations. It was not possible to evaluate the expression of Ki67 antigen in the second (larger) series of radical prostatectomy specimens. As most included specimens were obtained from different services in different cities, it was not possible to obtain patient follow-up data.
In conclusion, the present study found that Gleason 7 tumors with 4+3 status have higher proliferative indices. They also appear to be more infiltrative or locally aggressive than 3+4 tumors, which may be related to a higher frequency of extraprostatic extension and seminal vesicle invasion, despite no differences in the percentage of gland involvement by carcinoma or tumor volume. For those laboratories that employ partial samplings as the adopted method for processing radical prostatectomy specimens, the inclusion of the entire prostatic periphery and extraprostatic tissue is advised to avoid understaging due to missing evidence of extraprostatic extension.
This work was supported by a grant from CNPq (the National Council for Scientific and Technological Development; 474596/2012-5).
Epstein JI, Allsbrook WC Jr, Amin MB, Egevad LL; ISUP Grading Committee (2005) The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol 29: 1228-1242.
Billis A, Magna LA, Ferreira U (2003) Correlation between tumor extent in radical prostatectomies and preoperative PSA, histological grade, surgical margins, and extraprostatic extension: application of a new practical method for tumor extent evaluation. Int Braz J Urol 29: 113-119
Athanazio PR, Dos Santos AC, de Freitas LA, Athanazio DA (2014) A modified point count method as a practical approach to assess the tumor volume and the percent gland involvement by prostate carcinoma. Pathol Res Pract 210: 312-317.
Han M, Snow PB, Epstein JI, Chan TY, Jones KA, et al. (2000) A neural network predicts progression for men with gleason score 3+4 versus 4+3 tumors after radical prostatectomy. Urology 56: 994-999.
Sakr WA, Tefilli MV, Grignon DJ, Banerjee M, Dey J, et al. (2000) Gleason score 7 prostate cancer: a heterogeneous entity? Correlation with pathologic parameters and disease-free survival. Urology 56: 730-734.
Lau WK, Blute ML, Bostwick DG, Weaver AL, Sebo TJ, et al. (2001) Prognostic factors for survival of patients with pathological Gleason score 7 prostate cancer: differences in outcome between primary Gleason grades 3 and 4. J Urol 166: 1692-1697.
Herman CM, Kattan MW, Ohori M, Scardino PT, Wheeler TM (2001) Primary Gleason pattern as a predictor of disease progression in gleason score 7 prostate cancer: a multivariate analysis of 823 men treated with radical prostatectomy. Am J Surg Pathol 25: 657-660.
Hattab EM, Koch MO, Eble JN, Lin H, Cheng L (2006) Tertiary Gleason pattern 5 is a powerful predictor of biochemical relapse in patients with Gleason score 7 prostatic adenocarcinoma. J Urol 175: 1695-1699.
Sim HG, Telesca D, Culp SH, Ellis WJ, Lange PH, et al. (2008) Tertiary Gleason pattern 5 in Gleason 7 prostate cancer predicts pathological stage and biochemical recurrence. J Urol 179: 1775-1779.
Tollefson MK, Leibovich BC, Slezak JM, Zincke H, Blute ML (2006) Long-term prognostic significance of primary Gleason pattern in patients with Gleason score 7 prostate cancer: impact on prostate cancer specific survival. J Urol 175: 547-551.
Wright JL, Salinas CA, Lin DW, Kolb S, Koopmeiners J, et al. (2009) Prostate cancer specific mortality and Gleason 7 disease differences in prostate cancer outcomes between cases with Gleason 4 + 3 and Gleason 3 + 4 tumors in a population based cohort. J Urol 182: 2702-2707.
Merrick GS, Galbreath RW, Butler WM, Waller KE, Allen ZA, et al. (2007) Primary Gleason pattern does not impact survival after permanent interstitial brachytherapy for Gleason score 7 prostate cancer. Cancer 110: 289-296.
Kristiansen G (2012) Diagnostic and prognostic molecular biomarkers for prostate cancer. Histopathology 60: 125-141.
Fisher G, Yang ZH, Kudahetti S, Møller H, Scardino P, et al. (2013) Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort. Br J Cancer 108: 271-277.