Argyrophilic Nucleolar Organizer Regions and Alpha-fetoprotein in Adenomatous Hyperplasia in Human Cirrhotic Livers

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1 ANATOMIC PATHOLOGY Orgnal Artcle Argyrophlc Nucleolar Organzer Regons and Alpha-fetoproten n Adenomatous Hyperplasa n Human Crrhotc Lvers SHUICHI TERASAKI, M.D., TADASHI TERADA, M.D., YASUNI NAKANUMA, M.D., AKITAKA NONOMURA, M.D., MASASHI UNOURA, M.D., AND KENICHI KOBAYASHI, M.D. Recently, adenomatous hyperplasa (AH) of the lver has been suspected as a precancerous leson n human hepatocarcnogeness. The authors examned 75 cases of AH from 42 crrhotc lvers, usng stanng of argyrophlc nucleolar organzer regons (AgNORs). These reflect prolferatve cell actvty. Fndngs n AH were compared wth those seen n hepatocellular carcnoma (HCC) and other chronc lver dseases. Expresson of alphafetoproten (AFP) was also examned mmunohstochemcally. The authors classfed AH nto three types: ordnary (OAH), atypcal (AAH), and AH wth focal malgnancy (FM). OAH mples a lack of atypa; AAH represents AH wth structural and cellular atypa but wthout the features of overt carcnoma; and FM denotes AH wth foc of overt HCC. Forty of the 75 cases of AH were categorzed as OAH, 9 as AAH, and 6 as FM. The noncancerous areas of FM had features of AAH. The mean number of AgNORs n AH was ntermedate between that seen n crrhoss (2.93) and HCC (6.8) and showed a step-wse ncrease n the followng order: OAH (2.95), AAH (3.89), noncancerous areas n FM (4.58), and malgnant foc n FM (5.7). There was no sgnfcant dfference n AgNOR counts between OAH and crrhoss. AgNOR counts n AAH and FM were sgnfcantly hgher than those of OAH, and lower than those of HCC. AFP was postve n 2 of 25 HCCs and n malgnant foc of 3 FM lesons, but t was absent n OAH and AAH. These data suggest that OAH has a lmted capacty for prolferaton but that AAH and FM are much more replcatve. The latter two condtons are probably preneoplastc lesons or early forms of HCC. (Key words: Adenomatous hyperplasa; Argyrophlc nucleolar organzer regons (AgNORs); Hepatocarcnogeness) Am J Cln Pathol 99;95: The term "adenomatous hyperplasa" (AH) of the lver, n crrhoss have been gven varous names, such as as frst descrbed by Edmondson,' s ntended to denote "AH," ' 5 " "atypcal adenomatous hyperplasa," "adenomatod a szable hepatocellular nodule that occurs after acute or hyperplasa," 42 "adenomatous hyperplastc chronc lver njures; ths condton s especally assocated nodules," 2,6 "small mass lesons n crrhoss," 3 "hepatocellular wth crrhoss. Lesons of AH contan portal tracts havng pseudotumor," 4 and "macroregeneratve nod portal vens, hepatc arteres, and nterlobular ble ducts. ules." 3-7 " 9 However, the defnton and classfcaton of Recently, these nodular lesons, whch are clearly larger than other regeneratve lesons n crrhotc lvers, have been mplcated as putatve precancerous prolferatons n human hepatocarcnogeness. Ths contenton has been advanced because malgnant or atypcal hepatocellular foc are occasonally found wthn AH. 2 " Nodular lesons these tumefactons have been controversal n Japan. Moreover, ther bologc behavors, ncludng capactes for prolferatve actvty, reman unclear. Nucleolar organzer regons (NORs) represent loops of DNA that possess the genes for rbosomal RNA. NORs are assocated wth specfc protens. These are easly demonstrated by means of argyrophlc technques for NORs (AgNOR). In recent studes, AgNOR counts are From the Second Department of Pathology, Department of Dagnostc sgnfcantly greater n some malgnant neoplasms than Pathology, and Frst Department of Internal Medcne, Kanazawa Unversty School of Medcne, Kanazawa. Japan. Receved June 3, 99; receved revsed manuscrpt and accepted for publcaton August 28, 99. Address reprnt requests to Dr. Terada: Second Department of Pathology, Kanazawa Unversty School of Medcne, Kanazawa 92, Japan. n normal or bengn lesons. 5 " 2 Furthermore, the number of AgNORs has been found to correlate wth K67 postvty, 2 bromodeoxyurdne (BrdU) labelng ndces, 22 and certan phases of the cell cycle determned by flow cytometry All of the latter three ndcators represent cell Downloaded from on 6 February 28 85

2 TERASAKI ET AL. 85 AgNORs and AFP n Adenomatous Hyperplasa prolferatve actvty. Thus, the number of AgNORs n any tssue s thought to reflect the replcatve ablty of consttuent cells. In the current study, we classfed AH nto three types and examned the prolferatve characterstcs of each, by countng the number of AgNORs. We also assessed AH usng mmunohstochemcal stanng for alpha-fetoproten (AFP), whch s a marker of hepatocellular carcnoma (HCC). The results of these evaluatons were compared wth those seen n cases of HCC, crrhoss, and chronc actve hepatts and n normal lvers. Defnton of AH MATERIALS AND METHODS In ths study, AH was defned both grossly and mcroscopcally. It was manfest as a dstnct and expansve nodular leson, clearly beng larger than surroundng regeneratve nodules and larger than 8 mm n smallest dameter n crrhotc lvers (Fg. ). Mcroscopcally, AH was represented by hepatocellular nodules contanng anatomcally complete portal tracts. AHs consstng of hepatocytes wth some atypa, but not regarded as malgnant, were ncluded n ths study. Other examples wth foc of classc HCC were also ncluded. Hepatocellular nodules composed totally of carcnoma cells, and other nodular lesons such as focal nodular hyperplasa and lver cell adenoma were not ncluded n ths seres. Subjects and Tssue Samples Cases of AH were retrospectvely retreved from the surgcal and autopsyflesof our laboratores and afflated hosptals durng the perod A total of 75 cases were obtaned from 42 crrhotc lvers (8 from surgcally resected lver specmens and 24 from autopses). Normal lvers (22 cases) and examples of chronc actve hepatts (2 cases), crrhoss (24 cases), and HCC (25 cases) were also obtaned from our recent bopsy, surgcal, or autopsy fles. Normal lvers had been hstologcally dagnosed as "normal lver," showng "nonspecfc change," or showng "mld fatty change"; and bopses had been done because of clncal dagnoses nvolvng mld lver damage. All cases of chronc actve hepatts and crrhoss were devod of HCC. In ths seres, all HCCs were moderately or poorly dfferentated and corresponded to Edmondson-Stener's grade II, III, or IV. 25 The age and sex of the patents are shown n Table. Suspected causes of chronc lver dseases are also documented theren. All specmens were fxed n 4% formaldehyde soluton and embedded n paraffn. Sectons of 3 nm thckness were staned wth hematoxyln and eosn. &?$%**: l^e^^;-^ v'"*! *#7% ~vk. >. ~>f C XX.. <^ \. \ w~ * FIG. (left). Gross features of adenomatous hyperplasa. Three nodules of adenomatous hyperplasa (arrowheads) are seen. They have no true capsule and are clearly larger than the surroundng regeneratve nodules. FIG. 2 (rght). Ordnary adenomatous hyperplasa (lower). Arrowheads ndcate the boundary of adenomatous hyperplasa. Hepatocytes of ordnary adenomatous hyperplasa have no atypa and resemble hepatocytes of surroundng regeneratve nodules (upper). A portal tract (arrow) s present wthn the adenomatous hyperplasa. Hematoxyln and eosn (X5). Vol. 95 No. 6 Downloaded from on 6 February 28

3 852 ANATOMIC PATHOLOGY Orgnal Artcle TABLE MEAN NUMBER OF ARGYROPHILIC NUCLEOLAR ORGANIZER REGIONS Hstology No. of Subjects HB No. of Cases wth Forms Chronc Lver Dsease Ale Cry of PBC Age (mean ± SD) Sex (M:F) No. of Specmens Mean No. of AgNORs (mean ± SD) Normal lver CAH LC OAH AAH nmfoffm MFofFM HCC ± ± ± ± ± ± ± ± 8.6 2: : 3: 8:8 9:4 8: 8: 6: ± ± ± ± ± ± ± ±.4 HB = scrum HBsAg postve; Ale = alcohol: Cry = cryptogenc: PBC = prmary blary crrhoss; CAH = chronc actve hepatts: LC = lver crrhoss: OAH = ordnary adenomatous hyperplasa; AAH = atypcal adenomatous hyperplasa: nmf = nonmalgnant foc; FM = adenomatous hyperplasa wth focal malgnancy: MF = malgnant foc: HCC = hepatocellular carcnoma. Classfcaton of AH In ths study, AH was classfed nto three types, reflectng a slght modfcaton of a prevous report from our group 26 : ordnary AH (OAH), atypcal AH (AAH), and AH wth focal malgnancy (FM). OAH was composed of hepatocytes that dd not dffer n appearance from that of hepatocytes n surroundng crrhotc nodules; they showed no atypa (Fg. 2). AAH conssted of hepatocytes that dsplayed modest nuclear or structural atypa but were not overtly carcnomatous (Fg. 3). FM was defned as AH that contaned foc of HCC cells (Fg. 4). Based on these crtera, the 75 cases of AH were subdvded nto 4 of OAH, 9 of AAH, and 6 of FM. Noncancerous portons of all FM lesons showed features of AAH. Stanng for AgNORs Sectons 3 m n thckness were obtaned from formaln-fxed, paraffn-embedded blocks, and staned for AgNORs as descrbed by Crocker and Sklbeck. 7 Treatment wth 5% hyposulfte for 5 mnutes was used, to yeld permanent preparatons. 2 To dstngush hepatocytes from mesenchymal cells, sectons were counterstaned wth.3% methyl green. Countng Procedure for AgNORs In each case, hepatocytes, whch were selected randomly from lesonal areas, were examned lght mcroscopcally wth the use of a X ol-mmerson objectve lens and a X eye lens. The mean number of AgNORs was calculated. Only dstnct, dark brown, ndvdual ntranuclear dots were counted. Staned nucleol were scored as one dot. Immunohstochemcal Stanng for AFP Sectons of AH and HCC were mmunostaned for AFP, usng deparaffnzed sectons and the avdn-botn-peroxdase complex method of Hsu and assocates. 27 Brefly, sectons were pretreated wth methanolc hydrogen peroxde for 2 mnutes, and then wth normal goat serum (dluted :5 n phosphate-buffered salne) for 3 mnutes. Prmary rabbt antserum to human AFP (DAKO Corporaton, Santa Barbara, CA) was dluted : n phosphate-buffered salne, and appled to the sectons at 4 C overnght. The sectons were then treated wth botnylated goat antrabbt IgG (Vector Laboratores, Burlngame, CA) at a :25 dluton for 3 mnutes. Next, freshly prepared avdn-botn-peroxdase complex (Vectastan ABC Kt, Vector Laboratores) was appled to the sectons for 3 mnutes. The hstochemcal demonstraton of antbody bndng was accomplshed usng a mxture of hydrogen peroxde and 3,3'-damnobenzdne tetrahydrochlorde soluton (Sgma Chemcal Company, St. Lous, MO). Negatve controls were obtaned by substtutng phosphate-buffered salne and nonmmune rabbt serum for the prmary antbody. Statstcal Analyss Student's Mest and Fsher's exact test were used. AgNOR Stanng RESULTS Argyrophlc nucleolar organzer regons appeared as dark brown dots of varable sze, n hepatocellular nucle. In normal lver and cases of chronc actve hepatts, AgNORs were clear, large nclusons (-2 nm n dameter) (Fg. 5). Nucleol themselves also were staned. In crrhoss and AH, more numerous and smaller AgNORs were found (Fg. 5). In HCC, many more AgNORs were recognzed, and these showed more varable confguratons. They ranged from smple small dots to large rregular nclusons (Fg. 5). The mean numbers of AgNORs are A.J.C.P.-June 99 Downloaded from on 6 February 28

4 TERASAKI ET AL. 853 AgNORs and AFP n Adenomatous Hyperplasa,' ft ; t FIG. 3 (upper). Atypcal adenomatous hyperplasa (lower). Arrowheads ndcate the boundary of adenomatous hyperplasa. Hepatocytes of atypcal adenomatous hyperplasa have some atypa, ncludng small cells, mld nuclear hyperchromasa, and mld nuclear crowdng, and are dfferent n morphologc characterstcs from the hepatocytes of surroundng regeneratve nodules (upper). Hematoxyln and eosn (X5). V -a* *^ ** FIG. 4 (lower). Adenomatous hyperplasa wth focal malgnancy. A. Wthn the adenomatous hyperplasa, there s a focus of malgnancy (arrowheads). A portal tract (arrow) s present wthn the adenomatous hyperplasa. Hematoxyln and eosn (X5). B. Hgher magnfcaton of A The malgnant focus (upper) conssts of well-dfferentated trabecular hepatocellular carcnoma. The nonmalgnant areas (lower) show features of atypcal adenomatous hyperplasa. Arrowheads ndcate the boundary. Hematoxyln and eosn (X2). > w» : ' shown n Table and Fgure 6. The mean number of AgNORs was smlar n normal lvers (.62 ±.32) and cases of chronc actve hepatts (.76 ±.2). Crrhoss and OAH also showed smlar AgNOR values (2.93 ±.7 and 2.95 ±.67, respectvely) and had sgnfcantly hgher AgNOR counts than normal lver and cases of chronc actve hepatts. The mean AgNOR number of AAH (3.89 ±.4) and noncancerous areas of FM (4.58 ±.4) was sgnfcantly hgher than that of crrhoss and OAH. The mean AgNOR counts n HCC n FM (5.7 ±.34) and HCC (6.8 ±.4) were the hghest; they were sgnfcantly greater than those of AAH and noncancerous areas of FM. There was no sgnfcant dfference n the mean number of AgNORs n normal lver and examples of chronc actve hepatts; crrhoss and OAH; and malgnant foc n FM and HCC, (Table, Fg. 6) Vol. 95 No. 6 Downloaded from on 6 February 28

5 854 ANATOMIC PATHOLOGY Orgnal Artcle * =^ * t # < ** 5 H* *... # V. T> f ^ A Bj.'M ^ K*" "* *j ^jr% > wfc v»;' FIG. 5. Argyrophlc nucleolar organzer regons of normal lver (^4), lver crrhoss (B), atypcal adenomatous hyperplasa (C), and hepatocellular carcnoma (D). Argyrophlc nucleolar organzer regon stan (X6). A.J.C.P. -June 99 Downloaded from on 6 February 28

6 TERASAKI ET AL. 855 AgNORs and AFP n Adenomatous Hyperplasa s 2 s 6 E n.s. * * n.s. 4 4 : T.. ' 9 *. * n.s. I 7 Normal CAR LQ QAH AAH nmf MF lver of FM ot FM HCC A'-. ' '.. «~.w :^v3* t?.^f.*^ c-.r.v;n-; K t ^,^^^Mm^ FlG. 6 (upper). Scattergram showng the mean number of AgNORs of each lesonal area. CAH = chronc actve hepatts; LC = lver crrhoss; OAH = ordnary adenomatous hyperplasa; AAH = atypcal adenomatous hyperplasa; nmf = nonmalgnant foc: FM = AH wth focal malgnancy; MF = malgnant foc; HCC = hepatocellular carcnoma. * = P <.5; ** = P <.; n.s. = not sgnfcant. FlG. 7 (lower). Alpha-fetoproten n malgnant focus (arrowheads) n adenomatous hyperplasa. A (left). The area ndcated by arrowheads s a malgnant focus. Hematoxyln and eosn (X). B (rght) The malgnant area s postve for alpha-fetoproten (arrow). Immunostan (avdn-botnperoxdase method) for alpha-fetoproten counterstaned wth hematoxyln (X). B Immunohstochemcal Stanng for AFP Twelve of 25 HCCs (48.%) were postve for AFP. In AH, 3 of 6 cases of FM (8.8%) were postve for AFP (Fg. 7), but all examples of OAH and AAH were negatve for AFP (Table 2). Among the three cases of FM, AFP was present n malgnant foc n two nstances, and n malgnant and nonmalgnant areas n the remanng leson. DISCUSSION Recently, small space-occupyng lesons have been found n crrhotc lvers wth the use of advanced magng modaltes, and these commonly are resected surgcally wth a tentatve dagnoss of HCC. Some of these prolferatons are hstologcally smlar to adjacent regeneratve nodules; others do represent HCC, and stll others are equvocal as to ther bengnancy or malgnancy. Such Vol. 95 No. 6 Downloaded from on 6 February 28

7 856 ANATOMIC PATHOLOGY Orgnal Artcle TABLE 2. INCIDENCE OF POSITIVE STAINING FOR AFP Hstology OAH AAH nmfoffm MFofFM HCC Incdence of Postve Stan for AFP Postve/Total (%) /4 (.%) /9 (.%) /6(6.3%) 3/6(8.8%) 2/25(48.%) CAH = chronc actve hepatts: LC = lver crrhoss; OAH = ordnary adenomatous hyperplasa; AAH = atypcal adenomatous hyperplasa; nmf = nonmalgnant foc; FM = adenomatous hyperplasa wth focal malgnancy; MF = malgnant foc; HCC = hepatocellular carcnoma. nodular lesons that are not carcnomatous have been nterpreted as AH or other synonymous dagnoses " 4 n Japan. In recent studes, t has been suggested that AH may be a precancerous leson n hepatocarcnogeness. 2 " Our recent analyses 7 ' 8 found that ron-rch "macroregeneratve nodules" were frequently assocated wth "hyperplastc hepatocellular foc." Tsuda and colleagues reported a case of "atypcal adenomatous hyperplasa" contanng a small area of overt HCC. They also found that the latter leson and surroundng AAH showed the same restrcton pattern for ntegrated hepatts B vrus DNA, ndcatng that the two lesons were derved from an dentcal clone. Ths observaton was beleved to support the premalgnant nature of AH. However, the dagnoss of hepatc nodular lesons n ths context vares from one pathologst to another, and the exact bologc behavor of AH remans uncertan. In the current evaluaton, we defned AH as an expansle leson that was clearly larger than surroundng regeneratve nodules, larger than 8 mm n sze, and stuated n a crrhotc lver. Because the appearances of AH dffered from case to case and from area to area, and because some examples showed a varable degree of atypa, we concluded that there was more than one type of ths leson. 26 Thus, we classfed AH nto three types: OAH, AAH, and FM. Although, to us, ths subdvson seems to be reproducble and objectve, addtonal studes wll be necessary to valdate ths classfcaton. In recent studes, t has been reported that AgNORs provde useful data on malgnant tumors, preneoplastc lesons, and bengn prolferatons n many organs, ncludng the lver. 5 " 24 NORs are seen n metaphase n D and G group acrocentrc chromosomes 3, 4, 5, 2, and 22 n humans. 28 NOR-assocated protens may be related to RNA polymerase I, 29 B23, and C23, 3 " 32 and they may have regulatory functons n controllng the transcrpton of genes for rbosomal RNA. 33 Moreover, AgNOR counts have been shown recently to correlate wth proven prolferatve cell markers such as the BrdU labelng ndex, 22 K67-postvty, 2 and cell-knetc hstograms generated by flow cytometry. 23 ' 24 Thus, the number of NORs s beleved to reflect the replcatve actvty of cells. BrdU mmunostanng, K67 labelng, and flow cytometrc methods are dffcult to apply to paraffnembedded sectons. By contrast, the AgNOR method s readly applcable to such specmens. Analyses of AgNORs rarely have been reported n studes of hepatc dsorders. To the best of our knowledge, there have been only two studes of AgNORs on ths topc. Crocker and McGovern 6 reported that the mean number of AgNORs was sgnfcantly dfferent n normal, crrhotc, and carcnomatous lvers. The fgures they cted were smlar to our data. Tanaka and assocates 22 examned the mean number of AgNORs and the value of the BrdUlabelng ndex n several preneoplastc or neoplastc lesons n rat hepatocarcnogeness, and reported that the results of both assays correlated well and showed a step-wse ncrease n AgNOR counts or BrdU-labelng from normal lver to HCC. However, there have been no pror reports of AgNORs n AH. In ths study, the mean number of AgNORs n varous forms of AH was ntermedate between those of crrhoss and HCC, and showed a progressve ncrease n the followng order: OAH, AAH, noncancerous areas of FM, and malgnant foc n FM. The fndng that there was no dfference n the mean number of AgNORs n OAH and crrhoss may mply that OAH s smlar to regeneratve nodules not only hstologcally, 26 but also wth regard to prolferatve actvty. The mean numbers of AgNORs n AAH and n noncancerous areas of FM were sgnfcantly hgher than that of OAH and that of crrhoss and lower than that of HCC. Ths observaton suggests that AAH and the noncancerous areas of FM have more prolferatve capacty than OAH and crrhoss. AgNOR counts n malgnant foc of FM were hgher than those n AAH but smlar to those of HCC, supportng the contenton that hghly atypcal foc of FM are, n fact, carcnomatous. Alpha-fetoproten s a good marker of HCC. In our seres, 2 of 25 HCCs (48.%) were postve for ths oncofetal determnant. Ths rate of reactvty s slghtly hgher than those n other reports. 34 In AH, AFP was present n malgnant foc of three cases of FM but absent n all examples of OAH and AAH. These fndngs agan renforce the concluson that malgnant foc n AH are phenotypcally smlar or dentcal to overt HCC. In one case of FM, the noncancerous regon was also postve for AFP, perhaps sgnfyng that some hstologcally equvocal lesons may have undergone carcnomatous transformaton already. From the fndngs n ths study, t may be deduced that AH (partcularly AAH) s an mportant preneoplastc leson. In cases of AAH, subclones that are phenotypcally and genetcally dentcal to overt HCC may develop, gv- A.J.C.P.-June 99 Downloaded from on 6 February 28

8 TERASAKI ET AL. 857 AgNORs and AFP n Adenomatous Hyperplasa ngrseto FM. Some of the latter lesons may progress to become overt carcnomas. Acknowledgments. Ths study was supported n part by grants from the Japanese Motor Vehcle Fund and the Uehara Memoral Foundaton. REFERENCES. Edmondson HA. Bengn epthelal tumors and tumor-lke lesons of the lver. In: Okuda K, Peters RL, eds. Hepatocellular carcnoma. New York: John Wley and Sons, 976: Arakawa M, Kage M, Sughara S, Nakashma T, Suenaga M, Okuda K. Emergence of malgnant lesons wthn an adenomatous hyperplastc nodule n a crrhotc lver: observatons n fve cases. Gastroenterology 986;9: Furuya K, Nakamura M, Yamamoto Y, Toge K, Otsuka H. Macroregeneratve nodule of the lver. A clncopathologc study of 345 autopsy cases of chronc lver dsease. Cancer 988;6: Ohta G, Nakanuma Y. Comparatve study of the three nodular lesons n crrhoss. Adenomatod hyperplasa, adenomatod hyperplasa wth ntermedate leson, and small hepatocellular carcnoma. 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