Cellular features distinguishing benign and malignant phenotypes in prostatic biopsies

Human PATHOLOGY VOLUME 31

December 2000

NUMBER 12

Editorial Cellular Features Distinguishing Benign and Malignant Phenotypes in Prostatic Biopsies Increasing numbers of prostatic biopsies are being submitted for examination to exclude early prostate cancer. Morphological appearances, particularly of the epithelial component of these specimens, have emphasized the range of benign processes, which mimic or may be confused with adenocarcinoma. The diagnostic surgical pathologist is required to make three fundamental decisions when examining a prostatic needle core biopsy. First is to confirm whether a submitted tissue (meaning any contained epithelial structures) is of prostatic parenchymal origin, or of some other type. Common sources of nonparenchymal epithelia include Cowper’s glands, seminal vesicles, or verumontanum mucosa. Sampling Cowper’s glands only occurs in apical biopsies. Inclusions of rectal mucosa or transitional epithelium from the urinary bladder usually pose no diagnostic problems. Second, after confirming that contained epithelial structures are prostatic parenchymal, is to ascertain whether the glands are benign or malignant. This is more difficult and requires an appreciation of the wide spectrum of benign morphological appearances which can mimic prostatic carcinoma. While typical nodular hyperplasia poses little diagnostic difficulty, other patterns of epithelial proliferation share a variety of morphologic features with neoplastic processes, thus causing confusion or diagnostic problems. Postatrophic hyperplasia and basal cell hyperplasia are the most common and are viewed as variants of nodular hyperplasia. Cribriform hyperplasia is less common but also appears to be a variation of nodular hyperplasia. Atypical adenomatous hyperplasia is a controversial lesion which some consider to possibly be a precursor to adenocarcinoma, although at the present time most evidence is against this. Arguments as how best to designate such atypical appearances in surgical pathology reports have continued in recent issues of

Copyright © 2000 by W.B. Saunders Company doi:10.1053/hupa.2000.21282

Human Pathology.1,2 Sclerosing adenosis is a proliferation of both epithelial and stromal spindle cells with myoepithelial features. In different ways, each of these lesions exhibits particular morphologic features that may resemble prostatic adenocarcinoma, particularly when viewed “out of context” and in the absence of usual anatomical cues. These lesions have been the subject of a detailed appraisal by an expert panel of the World Health Organization.3 After excluding all other conditions, the third decision is to confirm the nature of any neoplastic glands along the spectrum from dysplasia to invasive malignancy with high metastatic potential. Morphological criteria for prostatic intraepithelial neoplasia (PIN) are well documented4 with recent recommendations that the qualifying terms “low” and “high” grade should be abandoned in favor of the simplified and unqualified terminology of “PIN” for that which was previously recognized as high-grade, since only this type exhibits any prognostic and diagnostic relevance.3 However, while PIN is the only morphological dysplasia currently recognized as a precursor of prostatic adenocarcinoma, it is unlikely to be the sole origin of this malignancy. PIN is associated with progressive abnormalities of phenotype and genotype that are intermediate between normal prostatic epithelium and cancer, indicating impairment of cell differentiation and regulatory control with advancing stages of prostatic carcinogenesis. Recent years have seen an increased search for markers that not only assist with the differential diagnosis of malignancy but also predict the phenotypic behaviour of individual malignancies. Use of monoclonal antibodies, such as 34␤E12 to high–molecular weight cytokeratins, has been of valuable assistance in the identification of basal cells and in distinguishing between benign and neoplastic glands.5 During prostatic carcinogenesis, there is progressive loss of markers of secretory differentiation, including PSA, cytoskeletal proteins including muscle-specific actin, S-100 protein,

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HUMAN PATHOLOGY

Volume 31, No. 12 (December 2000)

cytokeratins AE1/AE3, and vimentin, and a wide range of glycoproteins together with modulation of their oligosaccharide domains.6 While of undoubted assistance, such “negative” features that become abrogated during transition to malignancy are not as diagnostically valuable as “positive” markers that reveal novel aspects of neoplastic cells not exhibited by their non-neoplastic counterparts or progenitors. Conversely, other markers that show progressive increase include c-erbB-2 and bcl-2 oncoproteins, the ligand EGF and its receptor EGFr, type IV collagenase, Lewis V antigen, TGF-␣, apoptotic bodies, mitotic figures or Ki-67,7 PCNA, and microvessel density. In the current issue of Human Pathology, Cohen et al8 assess the value of prostate secretory granules (PSG) as an additional aid in the differential diagnosis of prostatic malignancy. Recent discovery of PSG to be a component of normal prostatic secretion prompted their evaluation of PSG as a potential adjunctive criterion to distinguish benign and malignant prostatic epithelia. In that study, PSG depletion was examined in 150 sequential core biopsy samples which ranged in epithelial phenotype from benign through dysplasia to Gleason grades 3 and 4 carcinoma. After processing in glutaraldehyde-based fixatives followed by H&E staining, PSG appeared as tiny, uniform, bright red granules within the cytoplasm of non-neoplastic secretory cells. In normal prostatic secretory epithelia, PSG are released luminally from the episodically disintegrating apocrine compartment derived from the apical one third of surface columnar cells.9,10 In contrast, the majority of dysplastic or malignant cells contained homogenous amphophilic cytoplasm, although there were some paler pink or clear cells containing vacuoles.9 Overall, PSG content was at least 75% depleted in 80% of carcinomas and in 63% of high-grade dysplasias such that no granules were seen at low-power magnification on routine H&E staining. Prostatic secretory granule content in high-grade dysplasia was intermediate between that of normal and malignant epithelium. Variation in PSG content was apparent within individual glands so that mildly dysplastic regions appeared almost normal with respect to PSG while adjacent severely dysplastic regions within the same gland were devoid of granules. The authors suggest that these morphological changes are supportive of dysplasia being a truly premalignant condition and composed of cells with varying degrees of deviation from normality. Thus, an initial sustained loss of PSG occurring in dysplasia is followed by a further reduction as tumor cells invade stroma. Incremental loss of PSG identifies and accentuates important biological stages of tumor progression and altered behavior. The biological basis of the observation that occasional carcinomas contained nearnormal numbers of PSG and that varying amounts of PSG occurred within 10% of cases is likely to reside in the diverse pathways initiating individual neoplasms. Sometimes, this occurrence related to local tumor grade while, in other cases, no clear distinction of grade was apparent. Enhanced staining contrast produced by glutaraldehyde-based fixatives clearly distinguished PSG-filled cytoplasm of benign cells from the amphophilic cytoplasm in malignancy and was strong and reliable be-

tween cases. Conversely, the benign-malignant distinction following formalin fixation was more randomly variable. The observed contrast between benign and malignant epithelium was especially prominent in small carcinoma foci, thus greatly assisting cancer recognition. Cohen et al8 propose several advantages to the use of glutaraldehyde-based tissue fixatives over conventional fixation and recommend a trial of glutaraldehyde-based fixatives as the optimum method of preserving prostatic needle biopsy specimens, despite possible confusion created by occasional cases of clear-cell carcinoma or PSG-rich carcinoma. After glutaraldehyde fixation, finely clumped nuclear chromatin of luminal cells differs from dark-stained basal cell nuclei and contrast with formalin-fixed tissue,11 clearly defining the biphasic prostate cell layers and thus providing adjunctive support for decisions made on the basis of cytokeratin immunostaining. This morphological advantage may be most useful in focal areas of active prostatitis where luminal secretory cells are atrophic such that PSG production is variably diminished. Finally, small numbers of isolated PSG identified at high magnification within many prostate cancers assist by distinguishing prostatic carcinomas from those of urothelial origin. This most recent report by Cohen et al8 raises several issues. The study provides additional corroborative information concerning the biology of prostatic epithelia, particularly modulation of normal biological processes in neoplasia, rather than identifying a unique diagnostic criterion. During the past decade, the search by many laboratories to find markers specific to malignancy has consistently failed to identify either proteins or gene sequences with exclusive pathognomonic properties. Rather, cumulative evidence indicates a dichotomy between processes that initiate neoplasia and those responsible for subsequent malignant behavior. Although the former appear, consistently, to be genetic in origin, all of the latter appear to be epigenetic and reflect sustained changes in homeostatic regulation. Thus, abnormal appearance and distribution of PSG clearly show changes to an important phenotypic property of prostatic secretory epithelial cells which, in prostate cancer, ranks alongside modulated normal processes such as novel expression of voltage-gated ion channels12 and abrogated expression of PKC-␤13 or HSP27.14 Initial loss of these latter proteins, in common with PSG, is not robust as a diagnostic criterion of malignancy when compared with a marker expressed only in neoplasia. However, their re-expression by some invasive prostatic carcinomas, correlated with aggressive behavior of individual tumors, emphasizes their value as markers of prognosis. With respect to specific genetic events promoting prostate cancer and subsequent metastasis, there is growing evidence that critical events in the progression of epithelia from normality through dysplasia to malignancy require loss of function of metastasis suppressor genes or enhanced activity of metastasis-promotor genes. Involvement of tumor suppressor genes RB and p53 during promotion of some prostate cancers is well recognized.15

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EDITORIAL (Christopher S. Foster)

However, mutation of p53 occurs as a late event, not an initiating event, in the majority of prostate cancers and does not appear to be directly responsible for promoting metastasis. Neither RB nor p53 are useful diagnostic markers to identify prostate cancer or to discriminate between benign and neoplastic prostatic epithelia. The first identified metastasis suppressor gene of prostate cancer is KAI-1.16 Located on human chromosome 11p11.2, KAI-1 encodes a transmembrane glycoprotein otherwise known as CD82. Expression of KAI-1 is directly activated by p53, and its down-regulation, either through p53 or alternative mechanisms, correlates with metastatic dissemination of prostatic and other cancers. However, as a diagnostic marker for use in conventionally-processed pathological tissues, generally-diminished or abrogated expression of this protein occurred variably in malignant or metastatic neoplasms,17 and, hence, it is not as powerful as a marker expressed only in malignancy or metastasis. Identification of genes or their products positively promoting progression of malignancy or metastasis is more technically challenging than is identification of suppressor genes. Nevertheless, several common features of genes capable of promoting progression of malignancy or dissemination of metastases are becoming apparent. First, all genes with these properties hitherto reported encode “normal” proteins such as ion channels12 or Ca2⫹-binding proteins.18 Second, all of the protein products encoded by these genes are regulators of intracellular homeostasis, either directly or indirectly as integral components of intracellular membranes.19 Third, because none of the genes encoding these proteins are mutated, their enhanced expression is probably epigenetic in origin. In conclusion, the current report by Cohen et al8 describes a novel phenotypic distinction between benign and malignant prostatic epithelial cells and recommends their technique as a routine diagnostic practice. This observation is both interesting and of importance because it highlights modulation of a normal biological process in malignancy. However, practical exploitation of this finding, as a routine adjunctive criterion of malignancy, is unlikely to be adopted as a widespread practice because tissue-processing to detect the PSG requires the use of a nonstandard fixation protocol, and the diagnostic end-point, with respect to identification of cancer, is a negative attribute. If current observations are representative of events occurring during the progression from normality through neoplasia to metastasis, then development of a one-step diagnostic “test” to confirm malignancy is not likely to be achieved until the biological and pathological nature of malignancy are fully appreciated. The corollary to the observation reported by Cohen et al8 emphasises 2 major dilemmas in cancer biology and diagnosis. First, neoplasia and malignancy are neither synonymous nor inevitably consequential. Second, invasion and metastasis (in contrast to neoplasia) appear to be a consequence of modulated normal homeostatic control, thus replicating properties of fetal cells in an embryo or normal migratory cells in a developmentally mature individual. If this hypothesis is correct, then the dis-

crete stages of dysplasia, neoplasia, malignancy, and metastasis must be defined by the functional interaction of several identifiable processes and not by a single unique parameter. The result of such vectorial analysis will provide the diagnostic pathologist with a robust criterion of malignancy and the oncologist with a tumor-specific target for therapeutic intervention. CHRISTOPHER S. FOSTER, MD, PHD, FRCPATH Department of Pathology University of Liverpool Liverpool, England REFERENCES 1. Murphy WM: ASAP is a bad idea. HUM PATHOL 30:601, 1999 2. Epstein JI: How should atypical prostate needle biopsies be reported? Controversies regarding the term “ASAP.” HUM PATHOL 30:1401-1402, 1999 3. Foster CS, Bostwick DG, Bonkhoff H, et al: Cellular and molecular pathology of prostate cancer precursors. Scand J Urol Nephrol Suppl 205:19-43, 2000 4. Bostwick DG, Sakr W: Prostatic intraepithelial neoplasia, in Foster CS, Bostwick DG (eds): Pathology of the Prostate. Philadelphia, PA, WB Saunders, 1997, pp 95-113 5. Brawer MK, Peehl DM, Stamey TA, et al: Keratin immunoreactivity in the benign and neoplastic human prostate. Cancer Res 45:3663-3667, 1985 6. Foster CS: Processing of N-linked oligosaccharide glycoproteins in human tumours. Br J Cancer 62:57-63, 1990 (suppl 10) 7. McLoughlin J, Foster CS, Price P, et al: Evaluation of Ki-67 monoclonal antibody as a prognostic indicator for prostatic carcinoma. Br J Urology 72:92-97, 1993 8. Cohen RJ, Beales MP, McNeal JE: Prostate secretory granules in normal and neoplastic prostate glands: A diagnostic aid to needle biopsy. HUM PATHOL 31:1515-1519, 2000 9. Cohen RJ, McNeal JE, Edgar SG, et al: Characterisation of cytoplasmic secretory granules (PSG), in prostatic epithelium and their transformation-induced loss in dysplasia and adenocarcinoma. HUM PATHOL 29:1488-1494, 1998 10. Cohen RJ, McNeal JE, Redmond SL, et al: Luminal contents of benign and malignant prostatic glands: Correspondence to altered secretory mechanisms. HUM PATHOL 31:94-100, 2000 11. Epstein JI: Diagnosing adenocarcinoma of the prostate on needle biopsy, in Silverberg SG (ed): Prostate Biopsy Interpretation (ed 2). Philadelphia, PA, Lippincott-Raven, 1995, pp 87-105 12. Smith P, Rhodes NP, Shortland AP, et al: Sodium channel protein expression enhances the invasiveness of rat and human prostate cancer cells. FEBS Lett 423:19-24, 1998 13. Cornford PA, Evans JD, Dodson AR, et al: Protein kinase C (PKC) isoenzyme patterns characteristically modulated in early prostate cancer. Am J Pathol 154:137-144, 1999 14. Cornford PA, Dodson AR, Parsons KF, et al: Heat shock protein (HSP) expression independently predicts clinical outcome in prostate cancer. Cancer Res 60:2000 (in press) 15. Foster CS, Cornford P, Forsyth L, et al: The cellular and molecular basis of prostate cancer. Br J Urology 83:171-194, 1999 16. Dong JT, Lamb WP, Rinker-Schaeffer CW, et al: KAI 1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 268:884-886, 1995 17. Ward JM, Konishi N, Oshima M, et al: Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines. Pathol Int 48:87-92, 1998 18. Ke Y, Jing C, Barraclough R, et al: Elevated expression of calcium-binding protein p9Ka is associated with increasing malignant characteristics of rat prostate carcinoma cells. Int J Cancer 71:832837, 1997 19. Jing C, Beesley C, Foster CS, et al: Identification of the messenger RNA for human cutaneous fatty acid-binding protein as a metastasis-inducer. Cancer Res 60:2390-2398, 2000

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