We selected 63 prostate tumors with Gleason's grade 6 (3+3), commonly showing both tubular and cribrous patterns. We compared in both patterns the expression of two of the most used biologic markers: PCNA and p53, with the aim to verify the validity of the Gleason's grading system to compare the morphologic grade with biologic aggressiveness and prognostic value. We did not find any statistical difference in the protein immunopositivity, indicating that both patterns could have identical biologic behaviour; then we confirmed the validity of Gleason's system for considering both tubular and cribrous patterns as an intermediate grade of tumoral differentiation. Moreover, we found a linear relationship between the increase of PCNA and the accumulation of mutated p53; this datum could confirm the hypothesis that p53 mutation is a late event in prostate carcinogenesis

ARAGONA, Federico

BARRESI, Elisabetta

CABIBI, Daniela

CAPPELLO, Francesco

FARINA, Felicia

MARTORANA, Anna

MELLONI, Darvinio

PALMA, Antonio

RAPPA, Francesca

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129©2003, European Journal of HistochemistryWe selected 63 prostate tumors with Gleason’s grade 6(3+3), commonly showing both tubular and cribrous pat-terns. We compared in both patterns the expression of two ofthe most used biologic markers: PCNA and p53, with the aimto verify the validity of the Gleason’s grading system to com-pare the morphologic grade with biologic aggressivenessand prognostic value. We did not find any statistical differ-ence in the protein immunopositivity, indicating that bothpatterns could have identical biologic behaviour; then weconfirmed the validity of Gleason’s system for consideringboth tubular and cribrous patterns as an intermediate gradeof tumoral differentiation. Moreover, we found a linear rela-tionship between the increase of PCNA and the accumula-tion of mutated p53; this datum could confirm the hypothe-sis that p53 mutation is a late event in prostate carcinogen-esis.Key words: proliferative index, tumoral differentiation,prostate cancerogenesis, tumoral prognosis.Correspondence: Francesco Cappello, M.D., via alla Falco-nara 120, 90136 Palermo, Italy. Fax.: international +39.091.6553518. E-mail: francapp@hotmail.comPaper accepted on October 5, 2002European Journal of Histochemistry2003; vol. 47 issue 2 [Apr-Jun]:129-132Biological aggressiveness evaluation  in prostate carcinomas:immunohistochemical analysis of PCNA and p53 in a seriesof Gleason 6 (3+3) adenocarcinomasF. Cappello,*° A. Palma,* A. Martorana,° F. Rappa,° D. Cabibi,° E. Barresi,° D. Melloni,^F. Farina,* F. Aragona°*Human Anatomy Section, Department of Experimental Medicine, University of Palermo; °Institute ofPathological Anatomy, University of Palermo; ^Department of Urological Sciences, University of Messina,ItalyProstate adenocarcinoma is one of the mostfrequent malignancy and the second leadingcause of cancer-related deaths in men inWestern Countries (Boring et al., 1992). It deter-mined a great focus on its pathogenesis as well asdiagnostic and prognostic criteria and therapeuticapproach.Gleason’s system is the most widely used gradingof prostate carcinoma, based on the tissue aspect atlow magnification (Gleason, 1977). The value isobtained identifying the predominant and the secondmost prevalent architectural pattern and assigning agrade from 1 to 5, with 1 being the most differenti-ated and 5 undifferentiated (Gleason, 1977).The main advantages of this system are that i) it iseasy to learn and apply and ii) it was well demon-strated its fairly well correlation with the prognosis(Kramer et al., 1980). The most frequent causes ofpitfall are i) the multifocality of the prostate cancerwith simultaneous variety of differentiation and ii)the tendency to undergraduate the bioptic materialwhen a minimal amount of tumor is present.The lat-ter can determine difficulty in valuing the variabilityof neoplastic gland size and shape, especially impor-tant to identify the Gleason’s grade 3. Moreover, thecombination of tumoral grade with other prognosticvariables to determine multiple prognostic indexesallows greater precision in predicting outcome(Kramer et al., 1980).Gleason 6 (3+3) is one of the most commonlyfound grade. It is characterised by both tubularand/or cribrous pattern of tumoral glands.Commonly, adenocarcinomas of other sites with atubular pattern are thought a more differentiatedform then the cribrous ones, considering the latter asan expression of an intermediate differentiation. Onthe contrary, the presence of one (or both) of thesepatterns in prostate adenocarcinoma, implies anintermediate grade of differentiation. Nevertheless,Original Paperto our knowledge, none study looked for the presenceof discrepancies between these two pattern in termsof biologic characterisation and behaviour.Here, we studied two important prognostic index-es in a large series of Gleason 6 (3+3) prostate ade-nocarcinomas: proliferating cell nuclear antigen(PCNA) and p53. PCNA is retained one of the mostimportant index to estimate the tumoral proliferateactivity (proliferating index, PI), while p53 is thewidest studied tumor suppressor gene, involved inregulation of cell cycle. The objective was to verifythe presence (or absence) of a discrepancy in theexpression of these antigens in both tubular andcribrous patterns.Materials and MethodsFiles of the Institute of Pathology of theUniversity of Palermo were searched for cases ofprostate carcinoma, Gleason 6 (3+3), during theperiod between 1st Jannuary 1998 and 31stDecember 2001. All specimens were been formalin-fixed and paraffin-embedded. We selected 63 cases,in which there were simultaneously present tubularand cribrous areas. Seven micrometers sections werestained with H&E for routinely diagnosis. Moreover,we performed an immunohistochemical analysis.We immunostained with avidin-biotin complex(LSAB2, DAKO), using primary antibodies againstPCNA (Clone PC10, DAKO, Cat. No. M 0879) andp53 (Clone DO-7, Dako, Cat. No. M 7001) respec-tively diluted at 1:50 and 1:40. Non-immune serumwas substituted for negative controls. Appropriatepositive controls were run concurrently for both anti-bodies tested. Aminoethilcarbazol (AEC chromogen,DAKO, Cat. No. K0697) was used as develop chro-mogen. A light nuclear counterstaining with hema-toxylin was finally performed.To better confirm the data, the immunostainingswere repeated three times on two sections for eachspecimen.Two independent observers carried out themicroscopical analysis.They valued: PCNA, countingthe percentage of positive tumoral cells in 10 HPF;p53, counting the number of tumoral glands in whichat least one cell expressing antigenic positivity waspresent in 10 HPF.We using the One way analysis of variance(ANOVA) to determine the presence of significantdifferences within the data. Differences between themeans of the values were regarded as significantwhen a p<0.05 was obtained. Moreover, we used the“r-Pearson” model to verify the presence of a linearrelationship between PCNA and p53 immunopositiv-ity (t-tab: 2,021).ResultsWe observed 63 cases of prostate adenocarcinomawith both cribrous and tubular pattern.Tumoral tis-sue showed closely packed small glands, usuallyirregularly separated, with poorly defined edge andsmoothly circumscribed masses of cribriform epithe-lium (Figure 1a). Moreover, tumoral cells present inthe glands differed from surrounding benign tissue inboth cytoplasmic or nuclear features. In particular,prominent nucleoli, nuclear enlargement, hyperchro-matic nuclei, amphophilic cytoplasm and mitotic fig-ures were common features (Figure 1b). In addition,basal cells were frequently absent and corpora ami-lacea were rare. Finally, high-grade PIN (prostaticintraepithelial neoplasia) foci were occasionallyfound in proximity of tumoral glands.Immunohistochemistry demonstrated the presenceof PCNA in all examined specimens (100%), whilep53 was present only in 40 of 63 cases (63,5%). Inparticular, the positivity of PCNA ranged between20% and 55% of the tumoral cells (in mean 35%)(Figure 1c), while p53 was expressed in a number oftumoral glands varying between 0% and 45% (inmean 14%) (Figure 1d). In addition, both PCNAand p53 positivity were occasionally found in high-grade PIN foci (0-5%), while normal glands showedrare and negligible positivity only to PCNA (0-1%).At the statistical analysis, no significative differ-ence resulted in positivity between these two mark-ers in both tubular and cribrous areas (p> 0.05). Inparticular, we found PCNA positivity in mean in34% of tubular area and 35% of cribrous areacells, while p53 was found in mean in 13% and 15%of tubular and cribrous glands, respectively.Moreover, a linear relationship between PCNA andp53 expression was found (t=2.235; t-tab=2.021;t>t-tab), examining the 40 cases in which bothmarkers were simultaneously present (Figure 2).DiscussionProliferating cell nuclear antigen (PCNA) is oneof the most studied proliferation-associated protein.It was demonstrated to be a strong sensitive indica-tor of cellular growth fraction (PI), because it isdetectable throughout most of the cell cycle, duringactive DNA synthesis or cell division (Hall et al.,1990). Moreover,Visakorpi reported a strong corre-lation between high level of PCNA staining and poor130F. Cappello et al. prognosis for patients with prostate carcinoma(Visakorpi, 1992). In addition, Montironi et al.(1993) showed that the number of PCNA-stainednuclei increased from small and large acinar pat-terns through cribriform to solid or trabecular pat-terns. Moreover, there was also a tendency for astronger staining at the invasive edge of the neoplas-tic area than in the central region.P53 is a tumor suppressor gene frequently alteredin malignant human tumors (Oren, 1992; Cappelloet al., 2001). In case of DNA damage, functional(wild) p53 may block cell-cycle progression in lateG1 phase or may trigger an intrinsic mechanism ofapoptosis (Levine et al., 1991, Cappello et al., inpress). By contrast, the abnormal p53 protein pro-duced by mutant gene is ineffective and more stablethan the wild type protein; it becomes less sensitiveto proteolysis and it tends to accumulate in thenucleus, so being easily detected by immunohisto-chemistry (Brunner et al., 1993). We used the mon-oclonal antibody clone DO-7 that recognised bothwild-type and mutant p53; however, because the wild131Original PaperFigure 1. Histologic and immunohis-tochemical aspects of studiedtumors. A: H&E stained sectionshowing both patterns: tubular(arrow) and cribrous (arrow-head),103. B. H&E showing the featuresof the tumoral cells, 403. C: PCNApositivity in both tubular (arrow)and cribrous (arrow-head) patterns,103. D: cribrous area showingnuclear positivity to p53. Inset:.p53positivity in tubular area, 253.Figure 2. Graphicshowing the linearrelationship betweenPCNA and p53 posi-tivity. 132type protein is rapidly degraded, its physiologic lev-els remain below the immunohistochemicaldetectably threshold. In this way, p53 immunoreac-tivity is likely to reflect p53 gene mutations(Papadopoulos et al., 1996). Numerous studies sug-gested the role of p53 mutation in the pathogenesisof prostate cancer, even if recent studies reportedconflicting results about the incidence of nuclearp53 accumulation (Samaan et al., 1993; Kallakuryet al., 1994; Fox et al., 1993; Shurbaji et al., 1995).In our study, PCNA resulted positive in all cases,with a mean percentage of 35% tumoral cells. Bycontrast, p53 was present in 40 of 63 cases, with amean value of 14% of tumoral glands. It shouldmean that the studied tumors had a moderate pro-liferation index and an intermediate differentiation.Moreover, we found a linear relationship between PIand p53 positivity. This datum indicated a correla-tion between the increase of proliferating index andthe accumulation of mutated p53. This is in agree-ment with Papadopoulos et al. (1996) that found ahighly significant correlation between p53 over-expression and tumor stage. They suggested themutation of the p53 gene was not an initial event inprostate carcinogenesis. It implied the existence of astrong correlation between p53 and tumor grade,suggesting also that aberrant p53 expression goesalong with a loss of differentiation. Indeed,Papadopoulos et al. (1996) found that tumors withincreased p53 positivity also exhibited a significanthigher proliferation index, this latter corroboratingthe former observation. In conclusion, in this workwe studied a series of prostate adenocarcinomaswith an intermediate grade of differentiation(Gleason 6: 3+3).There were simultaneously presentboth tubular and cribrous areas. Generally, the pres-ence of a tubular pattern in adenocarcinomas ofother sites than prostate addresses the diagnosistowards a well-differentiated tumor, while thecribrous aspect is commonly an index of intermedi-ate differentiation. Notwithstanding this, we did notfind any statistic difference between PCNA and p53expression in both tubular and cribrous patterns ofprostatic adenocarcinomas. Therefore, our resultsconfirmed the validity of the Gleason’s system toattribute an intermediate grade of differentiation atboth tubular and cribrous patterns.ReferencesBoring CC, Squires TS, Tong T. Cancer Statistic, 1992. Cancer J Clin1992;42:19-38.Bruner JM, Connelly JH, Saya H. p53 protein immunostaining in rou-tinely processed paraffin-embedded sections. Mod Pathol 1993;6:189-94.Cappello F, Barnes L. Synovial sarcoma and malignant mesothelioma ofthe pleura: review, differential diagnosis and possible role of apopto-sis. Pathology 2001;33:142-8.Cappello B, Bellafiore M, Palma A, Bucchieri F. Defective apoptosis andtumorigenesis. Role of p53 mutation and Fas-FasL system dysregula-tion. Eur J Histochem 2002;46:199-208.Fox SB, Persad RA, Royds J, Kone RN, Silcocks PB, Colins CC. p53 andc-myc expression in stage A1 prostatic adenocarcinoma: useful prog-nostic determinant? J Urol 1993;150:490-4.Gleason DF. Histologic grading and staging of prostatic carcinoma. In:Tannenbaum M, Ed. Urologic pathology: the prostate. Philadelphia:Lea & Febiger; 1977. p. 171-97.Hall PA, Levison DA, Woods AL,Yu CC, Kellock DB, Watkins JA, et al.Proliferation cell nuclear antigen (PCNA) immunolocalization inparaffin sections: an index of cell proliferation with evidence of dereg-ulated expression in some neoplasms. J Pathol 1990;162:285-94.Kallakury BVS, Figge J, Ross JS, Fisher HA, Figge HL, Jenninngs TA.Association of p53 immunoreactivity with high Gleason tumor gradein prostatic adenocarcinoma. Hum Pathol 1994;25:92-7.Kramer SA, Spahr J, Brendler CB, Glenn JF, Paulson DF. Experiencewith Gleason’s histopathologic grading in prostatic cancer. J Urol1980;124:223-30.Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor gene.Nature 1991;351:453-6.Montironi R, Galuzzi CM, Diamanti L, Giannulis I, Pisani E, ScarpelliM. Proliferating cell nuclear antigen (PCNA) in prostatic invasiveadenocarcinoma. Is the proliferation state in the marginal zone of thetumor higher than in the central part? Anticancer Res 1993;13: 129-32.Oren M. p53: the ultimate tumor suppressor gene? FASEB J 1992;6:3169-76.Papadopoulos I, Rudolph P, Wirth B, Weichert-Jacobsen K. p53 expres-sion, proliferation marker Ki-S5, DNA content and serum PSA: pos-sible biopotential markers in human prostatic cancer. Urology1996;48:261-8.Samaan S, Butler J, Morale B. p53 overaccumulation correlates withhigh tumor grade in prostatic adenocarcinoma. Am J Clin Pathol1993;99:336.Shurbaji MS, Kalbfleisch JH, Thurmond TS. Immunohistochemicaldetection of p53 protein as a prognostic indicator in prostate cancer.Hum Pathol 1995;26:106-9.Visakorpi T. Proliferative activity determined by DNA flow cytometryand proliferating cell nuclear antigen (PCNA) immunohistochemistryas a prognostic factor in prostatic carcinoma. J Pathol 1992;23:273-9.F. Cappello et al. 

Biological aggressiveness evaluation in prostate carcinomas: Immunohistochemical analysis of PCNA and p53 in a series of Gleason 6 (3+3) adenocarcinomas

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Biological aggressiveness evaluation in prostate carcinomas: Immunohistochemical analysis of PCNA and p53 in a series of Gleason 6 (3+3) adenocarcinomas

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