Frequencies of different nuclear morphological features in prostate adenocarcinoma

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Annals of Diagnostic Pathology 15 (2011) 414–421

Frequencies of different nuclear morphological features inprostate adenocarcinoma

Farhat Rashid, MBBS⁎, Anwar Ul Haque, DABPDepartment of Histopathology, Pakistan Institute of Medical Sciences, Islamabad, Pakistan

Received 26 May 2011; accepted 1 June 2011

Abstract Diagnosis of prostate adenocarcinoma is primarily based on morphological analysis. Nucleomegaly,

⁎ Corresponding aE-mail address: fr

1092-9134/$ – see frodoi:10.1016/j.anndiag

prominent nucleoli, and hyperchromasia constitute current nuclear diagnostic parameters but are seenin benign conditions, vary with Gleason grade, and pose diagnostic challenge in well-differentiatedtumors with accompanying inflammation or equivocal architectural features. In such cases, otherpleomorphic nuclear features such as variation in size and shape, irregular contours, nuclear membraneinfoldings, and nonuniform chromatin, which are not incorporated in formal evaluation, may provehelpful. Our aim was to study different nuclear morphological features of prostate adenocarcinoma(including currently practiced ones) and highlight their variation with Gleason grades. We examined84 cases of prostate adenocarcinoma using oil immersion microscopy where necessary. CommonestGleason pattern observed was grade 4a accounting for 42.8% of cases. Nuclear enlargement (moderateto marked in 93.8%), nucleolar enlargement (62.1%), and nonuniform chromatin distribution (100%)could serve as useful diagnostic features but did not vary with tumor differentiation. Pleomorphism(moderate in 58.6%), nuclear overlapping (62.8%), nuclear membrane infoldings (66.2%), andirregular contours (frequent in 94.5%) were significant diagnostic features that increased in frequencyand extent with increasing grade and could be used to differentiate low-grade from high-grade tumors.Worsening of nuclear morphology with advancing tumor grades indicated that nuclear anaplasiaaccompanies poor architectural differentiation. Coexistence of pale and dark nuclei signified variablechromatin density of no diagnostic significance.

Keywords: Prostate adenocarcinoma; Nuclear morphology; Gleason grades

1. Introduction

Prostate carcinoma is the second most commonmalignant tumor and sixth leading cause of cancer-relateddeaths in males, accounting for 903 500 new cases and258 400 deaths in 2008 [1]. Despite recent advances inmolecular genetics, light microscopic evaluation of archi-tectural and cytologic features on needle biopsy remains thegold standard for diagnosis of prostate adenocarcinoma[2-5]. However, approximately 18% to 28% [6-9] ofclinically significant cancers are missed on primary biopsy,and to overcome this problem, efforts have always been

uthor. Tel.: +92 512 202 [email protected] (F. Rashid).

nt matter © 2011 Elsevier Inc. All rights reserved.path.2011.06.002

made to study ongoing morphological changes and deviseancillary techniques that could help in early detection ofprostate carcinoma, differentiate it from benign mimics, andpredict its biologic behavior.

Nuclei have been center of research for decades. Nuclearatypia of malignancy is morphological expression ofongoing genetic and epigenetic changes in carcinogenesis[10-13]. Most of the published textbooks and journal articlesdescribe nuclear atypia of prostate adenocarcinoma in termsof prominent nucleoli, nucleomegaly, and hyperchromasia[2-5,8]. Although regarded as significant diagnostic features[2-5,8], these are nonspecific and often seen in many benignconditions such as atrophy, basal cell hyperplasia, and inreactive atypia caused by acute or chronic prostatitis,infarction, and irradiation [8,9,14]. In addition, appearanceof nucleoli, nuclear size, and chromasia are often influenced

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by differences in fixation, processing, section thickness, andstaining [3,4,8,15]. Diagnosing carcinoma alone on thesefeatures is an extremely difficult task especially whenarchitectural features are equivocal or difficult to assessbecause of scanty tissue in small biopsy specimens. In suchcases, analysis of additional features such as variation innuclear size and shape, irregular nuclear contours withindentations, undulations and folds of nuclear membrane,nonuniform chromatin texture, as well as changes in size,shape, and number of nucleoli, which are of great value indiagnostic evaluation of other tumors [16-20], are likely tohelp. Because prostate carcinoma shows variable degrees ofdifferentiation, another concern is variation of nuclearmorphology in different tumor grades. Many researchershave studied changes in nuclear size, shape, contours,chromatin, nuclear cytoplasmic ratio, and nucleoli and foundsignificant changes in different Gleason grades, but most ofthem used morphometry, [21-30], which is difficult to use inroutine practice because of complexity of image analysis.Some articles address morphological changes but consideronly few characteristics such as variation in nuclear size,shape, chromatin distribution, and nucleoli [9,31,32].

Our aim was to determine frequencies of different nuclearmorphological features in prostate adenocarcinoma andhighlight their variation with different Gleason grades.This knowledge will be extremely useful in the evaluationof low-grade tumors, tumors with equivocal architecturalfeatures, presence of confounding factors such as inflamma-tory atypia, and in very small biopsies and cytologypreparations devoid of sufficient architectural details.

2. Material and methods

This study was done at the Department of Histopathology,Pakistan Institute of Medical Sciences, after approval byregional research and training monitoring cell. FromSeptember 2010 to February 2011, 84 cases of prostateadenocarcinomawere selected by nonprobability consecutivesampling. These included 9 needle biopsies, 64 transurethralresections, and 11 prostatectomy specimens. Patients receiv-ing radiotherapy, hormones, or chemotherapy were excludedfrom the study to avoid confusion with radiation and drug-induced atypia. Each case was subcategorized into primary,secondary, and tertiary Gleason grades (where applicable).To reduce statistical errors, each tumor with similar primaryand secondary grades was analyzed as single case. Hema-toxylin and eosin–stained sections with a thickness of 3 to5 μm were first examined at low (×100) and high (×400)powers and then at oil immersion microscopy (×1000).Analysis of nuclear enlargement, pleomorphism (variation innuclear size and shape), nuclear overlapping, prominentnucleoli, chromatin appearance, chromatin distributionpattern, nuclear membrane infoldings and thickening, andnuclear contours was done in comparison with benign glandsin the specimens to reduce variability caused by differences in

fixation, section thickness, and staining. Nucleomegaly andpleomorphism were subcategorized as mild, moderate, ormarked; nuclear overlapping and membrane infoldings asabsent, focal, or frequent; nuclear contours as uniform ornonuniform; and chromatin appearance as (1) light or pale(hypochromatic and vesicular), (2) dark (dense hyperchro-matic and coarse chromatin), or (3) a combination of these.Nucleoli were designated as “prominent nucleoli” when seeneasily on low power and as “visible nucleoli” when seen onhigh power. Statistical analysis was done using SPSSpackage program (version 17; SPSS Inc, Chicago, IL, USA).

3. Results

Mean age was 71.1 ± 10.6 years with an age range of 51to 101 years. Commonest Gleason pattern observed wasgrade 4a accounting for 42.8% of cases. Nucleomegaly,pleomorphism, nonuniform chromatin distribution, andirregular nuclear contours were constant features in allcases, but their degree varied with Gleason grades. Normalprostate acinar cell nuclei were small, roughly equal to, orslightly larger than a small lymphocyte nucleus or a maturered blood cell. Nuclei were round or slightly angulated withregular contours, uniform dark chromatin, and occasionallyobserved tiny nucleoli. In prostate carcinoma, nuclei were 2or more times enlarged. Largest nuclei seen were evengreater than nuclei of vascular smooth muscle cells (Fig. 1).Nuclear enlargement and pleomorphism of mild to moderatedegree were seen in grade 1 and 2 tumors and of mild tomarked degree with bizarre nuclei in grade 3 to 5 tumors. Anexception was grade 4b tumors that showed hyperchromaticmildly enlarged nuclei with minimal pleomorphism butirregular borders (Figs. 2 and 3). Tumors of all gradesshowed irregularly distributed chromatin with alternatingareas of clearing, dense condensation with prominentchromocenters and vacuoles or bubbles. Contours wereirregular with bumps and indentations. However, this featurewas more pronounced in nuclei depicting hyperchromasia orcoarse chromatin. Nuclear overlapping was observed in 91(62.8%) cases, most of which belonged to grade 3 to 5category. Nuclear membranes were irregularly thickenedshowing infoldings in 96 (66.2%) cases (Fig. 4). Both low-and high-grade tumors showed prominent nucleoli, which inmany cases were multiple, eosinophilic, and often margin-ated (Fig. 5). Of the cases, 67.6% showed a combination of 2types of nuclei (Fig. 6); one type, “light or pale nuclei,” hadmarkedly enlarged round to ovoid nuclei showing minimalpleomorphism, vesicular irregularly distributed chromatin,large prominent and often multiple eosinophilic nucleoli,irregularly thickened nuclear membrane, irregular contours,and nuclear overlapping. Because of vesicular chromatin,these nuclei appeared pale with prominent eosinophilicnucleoli and fulfilled the commonly designated termprostate carcinoma looks back at you. These were observedin both low- and high-grade tumors. The other type of nuclei

Fig. 1. Nuclei in adenocarcinoma (A) are much greater than lymphocytes (arrow) and more or less equal to (B) nuclei of vascular smooth muscle cells (arrow).

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were “dark nuclei” that showed mild to marked pleomor-phism with bizarre forms, nucleomegaly, and nonuniformhyperchromatic or coarse clumpy chromatin with minuteirregular areas of clearing. Chromatin was dense enough toobscure separately visible nuclear membrane. However,nuclear contours were markedly irregular with indentationsand bumps. Nuclear overlapping was seen where nucleiwere clustered. Nucleoli were not visible even at oilimmersion microscopy. Dark nuclei were seen mostly incombination with vesicular nuclei in high-grade tumors(Tables 1 and 2). All cases showed extremely variablemorphology; the features observed were not seen in all cellsbut usually in most of the tumor cells.

Fig. 2. Compare small uniform nuclei of benign prostate acini (A and B) with gradpleomorphism.

4. Discussion

Nuclear atypia of malignancy is a known phenomenon. In1858, Rudolph Virchow published cellular pathologyexplaining the morphological characteristics of malignantcells [10]. Later, in 1860, Lionel S. Beale described nuclearsize and shape variations in cancer cells [13]. In 1953, Tottenet al first described prominent nucleoli as diagnostic featureof prostate carcinoma [15]. We observed nucleoli in 62% ofcases and macronucleoli in 37.2% of cases including 80% ofcases of grade 1 and 2 tumors, 21.6% of cases of grade 3tumors, and 42.4% of cases of high-grade tumors. Our resultswere nearly similar to a series of 300 carcinomas in which

e 2 tumour (C and D) showing prominent nuclear enlargement but minimal

Fig. 3. Pleomorphism in grade 3 tumour with perineural invasion (A), although nuclei appear smaller, few bizarre cells (arrows) are seen.Minimal (B) andmarked (C)pleomorphism in grade 4a tumour. Grade 4b tumour (D) showing small hyperchromatic nuclei and jagged contours highlighted on oil immersion microscopy (inset).

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24% lacked prominent nucleoli and another 25% had onlyrarely present prominent nucleoli [33]. In other studies,prominent nucleoli were observed in 20% [34] and 41.7% of

Fig. 4. Prostate carcinoma on oil immersion microscopy, nuclei show thickened mclearing and jagged nuclear contours (C and D).

low-grade tumors and in 81.1% of high-grade carcinomas[35]. Varma et al [15] observed prominent nucleoli in 94% ofcases of prostate carcinoma. Fine and Epstein [36] observed

embranes with infoldings (A and B), nonuniform chromatin with irregular

Fig. 5. Nucleoli in prostate carcinomamay appear central (A) or marginal (B), may show size variation (C) and may be partially obscured by dense chromatin (D).

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frequent nucleoli in 11% of Gleason 8 to 10 tumors. Incontrast to increased frequency of prominent nucleoli inhigh-grade tumors in these studies, we observed this featuremore frequently in low-grade tumors. This might be becauselow-grade tumors more frequently depicted clear vesicularchromatin in contrast to dense hyperchromasia of high-gradetumors, making nucleoli less evident. Other factors that couldaffect detection of nucleoli include poor fixation, thicksections, overstaining, lack of chromatin clearing, and actualabsence of nucleoli, for example, in foamy gland carcinoma,carcinoma with androgen deprivation treatment effect,and nucleolus-poor well-differentiated adenocarcinomas[3,4,8,15]. Number of nucleoli among different tumor gradeswas also variable; low-grade tumors showed single prominent

Fig. 6. Light and dark nuclei in prostate carcinoma (A and B). Light nuclei (white achromatin. Dark nuclei (black arrows) show dense chromatin and irregular contou

nucleolus,whereas high-grade tumors showed single aswell asmultiple nucleoli, which is similar to the observation by Fineand Epstein [36]. Prominent nucleoli in low-grade tumors incombination with enlarged minimally pleomorphic nuclei arelikely to mimic benign conditions such as inflammation, basalcell hyperplasia, and atrophy, although irregular chromatindistribution and jagged contours with membrane thickeningand infoldings are clue to diagnosis. Increase in number andsize of nucleoli in carcinoma is proposed to be amorphologicalexpression of increased ribosomal production induced byoverexpression of c-MYC, p27, retinoblastoma, p53, anddifferent growth factors [37,38].

We observed nuclear enlargement, pleomorphism (vari-ation in nuclear size and shape), nonuniform chromatin

rrows) show prominent nucleoli, thickened nuclear membranes and vesicularrs.

Table 1Nuclear morphological features and distribution of cases in different Gleason grades

All grades Grade 1 Grade 2 Grade 3 Grade 4a Grade 4b Grade 5

No. of cases (%) 145 1 (0.7) 4 (2.8) 31 (21.4) 62 (42.8) 10 (6.9) 37 (25.5)Nucleomegalya Mild 9 (6.2) 9 (90)

Moderate 74 (51.1) 1 4 (100) 29 (78.4) 32 (51.6) 1 (10) 13 (35.1)Marked 62 (42.7) 8 (21.6) 30 (48.4) 24 (64.9

Pleomorphisma Mild 13 (9) 1 1 (25) 1 (2.7) 10 (100)Moderate 85 (58.6) 3 (75) 35 (94.6) 45 (72.6) 8 (21.6)Marked 47 (32.4) 1 (2.7) 17 (27.4) 29 (78.4)

Nuclear overlappingb Absent 54 (37.2) 1 2 (50) 15 (40.5) 18 (29) 7 (70) 14 (37.8)Focal 42 (29) 2 (50) 12 (32.4) 19 (30.6) 1 (10) 8 (21.6)Frequent 49 (33.8) 10 (27) 25 (40.3) 2 (20) 15 (40.5)

Nucleoli Macronucleoli 54 (37.2) 1 3 (75) 8 (21.6) 29 (46.8) 13 (35.1)Visible nucleoli 36 (24.8) 1 (25) 11 (29.7) 18 (29) 10 (100) 8 (21.6)No nucleoli 55 (37.9) 18 (48.6) 15 (24.2) 16 (43.2)

Chromatin appearance Pale 10 (6.9) 1 (25) 2 (5.4) 6 (9.7) 10 (100) 1 (2.7)Dark 37 (25.5) 3 (75) 9 (24.3) 7 (11.3) 12 (32.4)Both 98 (67.6) 1 25 (70.3) 49 (79) 24 (64.9)

Chromatin distribution UniformNonuniform 145 (100) 1 4 31 62 10 37 (100)

Nuclear membrane thickening & infoldingb Absent 49 (33.8) 1 (25) 12 (32.4) 13 (21) 10 (100) 14 (37.8)Focal 50 (34.5) 1 3 (75) 14 (37.8) 23 (37.1) 13 (35.1)Frequent 46 (31.7) 11 (29.7) 26 (41.9) 10 (27)

Irregular contoursb Focal 8 (5.5) 1 (25) 2 (5.4) 2 (3.2) 3 (30)Frequent 137 (94.5) 1 3 (75) 35 (94.6) 60 (96.8) 7 (70) 37 (100)

a Category assigned on advanced degree (grade).b Category assigned on percentage of tumor cells.

able 2uclear morphology of prostate carcinoma cells in different Gleason grades

leasonrade

Nuclear morphology

rade 1 Mild to moderately enlarged nuclei with minimalpleomorphism, prominent nucleoli, nonuniform chromatin,focal nuclear membrane folds, and frequently irregularcontours. Chromatin shows a combination of vesicularand hyperchromasia.

rade 2 All features of grade 1 but pleomorphism of mild to moderatedegree may be seen.

rade 3 Mild to marked nucleomegaly, usually moderate degree ofpleomorphism, enlarged nucleoli, focal nuclear overlapping,membrane thickening and infolding, frequent irregularity ofnuclear contours, and nonuniform pale or dark chromatin.

rade 4a Moderate to marked nuclear enlargement and pleomorphism,enlarged nucleoli, frequent nuclear overlapping, membraneinfoldings and jagged contours, and a combination of pale anddark nuclei with nonuniform chromatin distribution.

rade 4b Mild to moderately enlarged minimally pleomorphic,hyperchromatic nuclei with irregular chromatin distributionand jagged contours. Nuclear overlapping, prominent nucleoli,and membrane infoldings are not seen.

rade 5 Moderate to marked nuclear enlargement and pleomorphism,frequent jagged contours, and a combination of pale and darknuclei with nonuniform chromatin distribution. Prominentnucleoli, nuclear overlapping, and membrane infoldings maybe seen in vesicular nuclei (invisible in nuclei with densechromatin in the same tumor).

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distribution, and irregular nuclear contours in all tumorgrades. Nuclear enlargement was moderate in 51.1% andmarked in 42.7% cases. Thorson et al [39] observed nuclearenlargement in 96%. Atypia and irregular contours increasedin frequency and extent with increasing grade. Grade 1 and 2tumors showed mild to moderate variation in size and shape,grade 3 and 4 tumors showed moderate pleomorphism inmost cases (94.6% in grade 3 and 72.6% in grade 4), andgrade 5 tumors mostly showed marked pleomorphism(78.4%). Mostofi et al [9,31] described prostate carcinomacells as having minimal variation in nuclear size and shape.We found such features only in light or pale nuclei in grade 1to 3 tumors. Our observation is more in accordance withfindings of Bocking et al [30]. They observed mild anaplasiain 52.6% and moderate anaplasia in 47.4% of well-differentiated adenocarcinoma and mostly marked (72%)but sometimes moderate anaplasia (27%) in solid, undiffer-entiated carcinomas. Helpap [27] also observed mildanaplasia in low-grade tumors andmarked in undifferentiatedtumors. In our study, high-grade tumors showed morefrequent irregularity of nuclear contours, which is inaccordance with observation of Ross et al [29]. They foundround nuclei in well-differentiated cancers and irregularnuclear contours in high-grade tumors. Finding of increasingdegree of pleomorphism, frequent nuclear overlapping,nuclear membrane infoldings, and irregular nuclear contoursreflect lack of nuclear differentiation in accordance with poorarchitectural differentiation with increasing Gleason grades.

We noticed nuclear hyperchromasia in 93.1% of cases.Hyperchromatic nuclei in low-grade and grade 4b tumors

show minimal pleomorphism and irregular chromatindistribution, and jagged nuclear contours are significantclues to malignancy in such cases. Artifactual hyperchro-

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matism could be caused by compression, cauterization, oroverstaining, but in such cases, nuclei appear crushed andcytoplasm is fragmented.

Nuclear membranes showed irregular thickenings andinfoldings in 66.2% cases in both low- and high-gradetumors where chromatin appeared vesicular or hypochro-matic and became inconspicuous focally in nuclei withchromatin condensation in the same tumor. Irregularmembrane thickening occurs because of peripheral conden-sation of chromatin in a haphazard manner. Infoldings andirregularity of nuclear envelop may develop dynamicallyduring interphase after oncogene expression in clonalevolution of prostate cancer and reflect cytoskeletal forcesexerted on the nuclear envelop (NE) during active celllocomotion. Furthermore, it could promote chromosomalinstability and impact replication competence, transcriptionalprogramming, and nuclear pore function [38].

5. Conclusions

Pleomorphism, nonuniform chromatin distribution, andirregular nuclear contours could serve as useful diagnosticfeatures in the evaluation of prostate adenocarcinoma inaddition to currently recommended prominent nucleoli,nucleomegaly, and hyperchromasia. Nuclear overlappingand nuclear membrane irregularities could also be helpful.Coexistence of pale vesicular/smudgy and dark hyperchro-matic nuclei signifies variable chromatin appearance of nodiagnostic significance.

Nuclear enlargement, prominent nucleoli, and nonuni-form chromatin distribution are important diagnostic featuresbut do not vary with Gleason grades, whereas pleomor-phism, nuclear overlapping, nuclear membrane infoldings,and irregular contours increased in frequency and extent withincreasing tumor grades. Moderate to marked variation innuclear size and shape with frequent overlapping, membraneinfoldings, and irregular contours signify high-grade tumorsin contrast to mild to moderate variation in nuclear size andshape with focal nuclear overlapping, membrane infoldings,and irregular contours in low-grade tumors.

Acknowledgment

Our sincere gratitude to Dr. Noor Khan Lakhnana for hiskind support in data collection.

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