p53 and erbB-2 are not associated in matched cases of primary …downloads.hindawi.com/journals/dm/2003/108643.pdf · 2019-08-01 · Disease Markers 19 (2003,2004) 11–17 11 IOS

Disease Markers 19 (2003,2004) 11–17 11IOS Press

p53 and erbB-2 are not associated in matchedcases of primary and metastatic ovariancarcinomas

Cristina Rodrıguez-Burforda, David C. Chhienga, Cecil R. Stockarda, Marc J. Kleinbergb,Mack N. Barnesb, Edward E. Partridgeb, Heidi L. Weissc and William E. Grizzlea,∗aDepartment of Pathology,bDepartment of Obstetrics and Gynecology, andcComprehensive Cancer CenterBiostatistics Unit, The University of Alabama at Birmingham, Birmingham, AL 35294, USA

Abstract. Ovarian cancer has a high mortality rate largely due to the limited number of ovarian carcinomas detected at anearly stage. Understanding the molecular changes occurring during the progression of ovarian carcinoma would aid in thedevelopment of therapies that may inhibit or target metastasis. Primary and metastatic lesions from 54 and 40 patients withadvanced ovarian carcinoma, respectively (including matched primary and metastatic lesions from 30 patients) were evaluated fornuclear accumulation of p53 (clone BP53-12-1) and cytoplasmic and membranous immunostaining of p185 erbB-2 (clone 3B5) byimmunohistochemistry. No differences in the immunostaining of p53 and p185erbB-2 (cytoplasm or membrane) were observedbetween primary and metastatic lesions of the matched cases. Similarly, no differences in the proportion of positive cases of p53between primary and metastatic lesions of the matched cases was observed. Thus, novel therapies that target p53 or p185erbB-2can utilize specimens from either primary or metastatic lesions to characterize these targets prior to therapy. Spearman correlationsbetween p53 and p185erbB-2 (cytoplasm or membrane) immunohistochemistry scores were insignificant for the matched cases,all primary lesions, and all metastatic lesions. Also, no significant associations occurred between nuclear accumulation of p53(positive versus negative) and phenotypic expression of p185erbB-2 (cytoplasm or membrane) immunostaining scores for thematched cases, all primary lesions, and all metastatic lesions. Thus, the nuclear accumulation of p53 and immunostaining ofp185erbB-2 in the cytoplasm or on the cellular membranes are independent.

Keywords: Ovarian carcinoma, molecular markers, p53, and p185 erbB-2

1. Introduction

Ovarian carcinoma remains as the leading cause ofdeath among gynecologic malignancies in the WesternWorld [8]. Among ovarian carcinomas, tumors hy-pothesized to arise from the surface epithelium of theovary are the most common, consisting of 80–90% ofcases [18]. Due to a lack of specific symptoms, themajority of ovarian carcinomas usually presents at anadvanced stage and thus, lead to poor survival [13]. To

∗Address for correspondence: W.E. Grizzle, M.D., Ph.D., Dept.of Pathology, The University of Alabama at Birmingham, AL 35294,USA. E-mail: [email protected].

date, the molecular mechanisms of ovarian carcinomaare poorly understood. The prognosis of a patient islargely dependent on morphologic criteria. No tissuespecific markers have been identified to determine thegenetic etiology of ovarian cancer.

A primary objective of ovarian carcinogenesis stud-ies has been to characterize biomarkers useful for prog-nosis of ovarian cancer. These biomarkers includeoncogenes and tumor suppressor genes. The tumorsuppressor gene, p53, is commonly mutated in cancer.Overexpression of p53 has been observed in 50% oflung, colon, and ovarian cancers [19]. Mutations in p53have been described as a late genetic change in ovar-ian carcinogenesis [16,17]. The p185 erbB-2 (HER-2/neu) proto-oncogene encodes a 185-kDa transmem-

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12 C. Rodrıguez-Burford / p53 and erbB-2 are not associated in matched cases

brane protein that has sequence homology with epider-mal growth factor receptor may also play an impor-tant role in ovarian carcinogenesis [20]. The oncogeneproduct of p185 erbB-2 is expressed by most ovariancancers and overexpressed in approximately 15–30%of these cancers [7]. Overexpression of p185erbB-2 atthe cellular membrane has been associated with poorsurvival [2,20,24].

Studies have reported overexpression of p185erbB-2together with p53 nuclear accumulation in ovarian car-cinomas [4,7,12,26]. Some of these studies have ob-served a difference between benign and malignant ovar-ian tumors such that no or low expression of p53 andp185 erbB-2 are observed in benign lesions comparedwith nuclear accumulation of p53 and overexpressionof erbB-2 in malignant lesions of the ovary [1,11,12,26]. However, a limited number of studies have beenpublished focusing on the relationship between p53 andp185 erbB-2 in individual tumors [2,3,5,6,25]. Thus,we are interested in determining if any differences inexpression of p53 and p185 erbB-2 are observed be-tween primary and metastatic lesions of the ovary. Un-derstanding the molecular changes occurring duringovarian carcinogenesis would aid in the development oftargeted interventions that may inhibit or target metas-tasis. In this analysis, primary and metastatic cases,including matched cases of primary and metastatic le-sions were evaluated for nuclear accumulation of p53and cytoplasmic and membranous immunostaining ofp185 erbB-2 in patients with advanced chemotherapyresistant ovarian carcinoma.

2. Materials and methods

Paraffin-embedded tissues of patients who under-went surgery for ovarian epithelial neoplasia werecollected from hospitals throughout the SoutheasternUnited States during 1993–1999 as part of therapeu-tic trials at the University of Alabama at Birmingham.Thus, our study included only patients with advancedovarian carcinoma who ultimately developed peritonealmetastasis and failed conventional chemotherapy. Allpatients were treated with surgical debulking and plat-inum based chemotherapy from 1993 to 1999. A totalof 54 primary and 40 metastatic cases, including 30matched primary and metastatic cases, were collected.

2.1. Immunohistochemistry

Paraffin-embedded tissues were sectioned at 5µmand attached to SuperFrost/Plus slides (Fisher Scien-tific, Norcross, GA) by heating at 58◦C for one hour.Immunohistochemistry was performed as previouslydescribed [9,10,15]. Tissue sections were deparaf-finized in xylene (three changes, 5 minutes each) andsubsequently rehydrated in sequential ethanols for 5minutes with one change each of absolute,95% ethanol,and 70% ethanol. The sections were then transferredto a Tris-buffer bath (0.05M Tris base, 0.15 NaCl,0.0002% Triton X-100, pH 7.6).

Each section was treated with an aqueous solutionof 3% H2O2 for 5 minutes in order to quench endoge-nous peroxidase activity. Each section was then incu-bated with 1% goat serum at room temperature for 20minutes in order to reduce non-specific immunostain-ing. A monoclonal antibody specific for p53 (cloneBP53 12.1, Biogenex, San Ramon, CA) and a mono-clonal antibody specific for p185 erbB-2 (clone, 3B5,Oncogene Science, Cambridge, MA) were used at di-lutions/concentrations of 1:400 and 0.25 ug/ml, respec-tively. Primary antibodies were incubated for 1 hr atroom temperature. Negative controls remained in goatserum (1%) at room temperature for 1 hr.

Primary antibodies were detected using a multi-species detection system from Biogenex (San Ramon,CA) or subsequently Signet Laboratories, Inc. (Ded-ham, MA). The sections were exposed to an anti-mouse and anti-rabbit antibody for 20 minutes and aperoxidase-labeled streptavidin was added for 20 min-utes. A diaminobenzidine tetrachloride (DAB) supersensitive substrate kit (Biogenex, San Ramon, CA) wasused to visualize the antibody-antigen complex. Theneach section was counterstainedusing hematoxylin, de-hydrated using graded alcohols, and soaked in xyleneprior to attaching coverslips with permount.

The slides were examined by at least two investiga-tors (CR-B, DCC, and WEG), each of whom estimatedthe intensity and extent of immunostaining. For p53and p185 erbB-2, tumor cells were classified with re-spect to percentage of cells stained at each intensityfrom 0 (no staining) to 4 (highest intensity staining) [9].If the staining intensity was no greater than the negativecontrols, then a score of zero was assigned for a spec-imen. A cytoplasmic and membrane immunostainingscore was determined for p185 erbB-2 whereas a nu-clear immunostaining score was determined for p53.The percentage of nuclei stained positive for p53 wasalso evaluated and a percentage of 10% or greater wasconsidered positive p53 staining.

C. Rodrıguez-Burford / p53 and erbB-2 are not associated in matched cases 13

2.2. Statistical analysis

Statistical analyses were conducted on the matchedcases. The Wilcoxon Signed- Rank test was used to testif there was a difference between p53 immunostainingscores in the primary lesions versus the metastatic le-sions in the matched cases (n = 30). Similarly, theWilcoxon Signed-Rank test was used to test if there wasa difference between p185 erbB-2 (membrane and cyto-plasm) primary immunostaining scores and p185 erbB-2 (membrane and cytoplasm) metastatic immunostain-ing scores in the matched cases (n = 26). A Spearmancorrelation was utilized to determine if any associationsbetween p53 and p185 erbB-2 (cytoplasm or mem-brane) immunostaining scores in the matched caseswere observed. The association of p185 erbB-2 (cyto-plasm or membrane) and p53 positivity (with positiv-ity defined as> 10%) between primary and metastaticlesions in the matched cases was determined using theWilcoxon Two-Sample test. The McNemar test wasused to test if a change in the percentage of p53 nu-clear accumulation was significantly different betweenprimary and metastatic lesions in the matched cases.

Statistical analyses were also conducted with all pri-mary lesions. The primary lesions (n = 54) includedprimary lesions with matched metastatic lesions as wellas primary lesions without matched metastatic lesions.A Spearman correlation was used to determine if anyassociations between p53 and p185 erbB-2 (cytoplasmor membrane) immunostaining scores were observedin all primary lesions. The Wilcoxon Two-Sample testwas used to determine any associations between p53positivity and erbB-2 (cytoplasm or membrane) im-munostaining scores in all of the primary lesions. Thesame statistical analyses that were conducted on theprimary lesions were performed with the immunohis-tochemistry scores for the metastatic lesions. The to-tal number of metastatic lesions (n = 40) includedmetastatic lesions with matched primary lesions as wellas metastatic lesions without matched primary lesions.

Descriptive statistics were determined for age, race,parity, menopausal status, hormone replacement ther-apy, stage, histological classification of tumors, and ra-diation treatment using SAS statistical software [23].Kaplan-Meier survival curves were estimated for allpatients overall as well as stratified by grade, stage, hor-mone replacement therapy treatment, and menopausalstatus. A percentage of nuclear accumulation of p53 of10% or greater was considered positive. Immunohisto-chemistry scores of p185 erbB-2 for the membrane andthe cytoplasm that were above the median were consid-

ered positive. Survival curves between p53 positive vs.p53 negative patients and between p185 erbB-2 posi-tive vs. p185 erbB-2 negative patients were also esti-mated for all matched, primary and metastatic lesions.The log-rank test was used to determine if there werestatistically significant differences in survival curvesbetween the clinical variables and levels of the twomolecular markers.

3. Results

Table 1 includes the descriptive statistics for grade,stage, and histologic subtype of ovarian carcinoma le-sions included in this study. Overall, the majority ofthe patients had high grade (grade 3) and stage III ovar-ian carcinomas. The largest proportion of the ovariancarcinomas was of the serous histologic subtype. Theaverage age of each patient was 57.4 and the averageparity was 2.5. Sixty-six patients were Caucasian and2 patients were black. Forty-three patients were ofpost-menopausal status, whereas 13 patients were ofpre-menopausal status. Thirty-seven patients were noton hormone replacement therapy and 19 patients werereceiving hormone replacement therapy.

A total of 54 primary and 40 metastatic lesions,including 30 primary and matched metastatic lesionswere identified. Each lesion was evaluated for nuclearaccumulation of p53 and cytoplasmic and membra-nous immunostaining of p185 erbB-2 (Fig. 1). For p53evaluations, immunostaining scores as well as percent-age positivity was determined. Only immunostainingscores were determined for p185 erbB-2.

For the matched cases, no significant differences inimmunohistochemistry scores of p53 (p = 0.6018),p185 erbB-2 immunostaining in the cytoplasm (p =0.6210), and p185 erbB-2 immunostaining in the mem-brane (p = 0.7910) were observed between primaryand metastatic lesions. The mean differences and stan-dard error means of the immunohistochemistry scoresbetween primary and metastatic lesions of the matchedcases for p185 erbB-2 membrane and p185 erbB-2cytoplasm were−0.07 ± 0.11, and0.03 ± 0.12, re-spectively (Fig. 2). No difference was also observedwith the proportion of positive cases of p53 betweenprimary and metastatic lesions of the matched cases(p = 0.1797) (Table 2). Spearman correlations be-tween p53 and p185 erbB-2 (cytoplasm and membrane)immunohistochemistry scores were insignificant for thematched cases, all primary lesions, and all metastaticlesions. Similarly, no significant associations occurred


Table 1Descriptive statistics for grade, stage, and histologic subtype of ovariancarcinomas

Gradea Number of patients Percentage of patients

1 5 9.82 14 27.453 32 62.75Tumor stageb Number of patients Percentage of patientsI 1 1.43IIB 2 2.86IIC 2 2.86III 1 1.43IIIB 5 7.14IIIC 52 74.29IV 7 10.00Histologic subtype Number of patients Percentage of patientsSerous 48 67.60Mucinous 2 2.81Endometrial 8 11.27Clear cell 1 1.41Mixed histologic type 3 4.23Adenocarcinoma NOSc 9 12.68aData for grade of the ovarian carcinoma was unavailable for 20 patients.bData for stage of the ovarian carcinoma was unavailable for 1 patient.cAdenocarcinoma not otherwise specified.

Table 2Proportion of cases with nuclear accumulation of p53 (p53NAC) and negativefor p53 (p53NEG) among the matched primary and metastatic lesionsa

Case description Number of cases

Primary Tumor+ Metastatic Tumor, both p53NAC 11Primary Tumor+ Metastatic Tumor, both p53NEG 14Primary Tumor p53NEG+ Metastatic Tumor p53NAC 1Primary Tumor p53NAC+ Metastatic Tumor p53NEG 4aA 10% or greater cut-off value was considered positive for p53.

between p53 positivity (positive versus negative) anderbB-2 (cytoplasm and membrane) immunohistochem-istry scores for the matched cases, all primary lesions,and all metastatic lesions.

Primary lesions (n = 45) were evaluated for pres-ence of adjacent uninvolved tissue. A total of 24 casescontained normal appearing uninvolved tissue. Nu-clear accumulation of p53 as well as cytoplasmic andmembranous immunostaining of p185 erbB-2 was eval-uated. The average p53 immunohistochemistry score(n = 21) was 0.0. The average immunohistochemistryscore for cytoplasmic and membranous expression ofp185 erbB-2 was 0.71 (n = 19) and 0.095 (n = 19),respectively.

Survival estimates indicated that the median sur-vival time was 36 months. Interestingly, there was nosignificant difference in survival time based on grade(p = 0.816), stage (p = 0.5181), menopausal sta-tus (p = 0.5433), and hormone replacement therapytreatment (p = 0.4566). Survival estimates basedon the nuclear accumulation of p53 and the cytoplas-

mic and membranous staining of p185 erbB-2 werealso analyzed. Based on p53 positivity (positive ver-sus negative), no significant differences were observedin survival time for primary lesions of matched cases(p = 0.2772), metastatic lesions of matched cases(p = 0.3460), all primary lesions (p = 0.7323), and allmetastatic lesions (p = 0.6012). For survival estimatesof p185 erbB-2, immunohistochemistry scores of im-munostaining in the membrane and of immunostain-ing in the cytoplasm that were above the median wereconsidered positive. Based on the immunostaining ofp185 erbB-2 in the membrane, no significant differ-ences were observed with survival time for primary le-sions of matched cases (p = 0.7661), metastatic lesionsof matched cases (p = 0.7117), all primary lesions(p = 0.4631), and all metastatic lesions (p = 0.5023).Similarly, no significant differences were found withimmunostaining scores of erbB-2 in the cytoplasm andsurvival time for primary lesions of matched cases(p = 0.8589), metastatic lesions of matched cases


Fig. 1. Immunohistochemical staining of p185 erbB-2 on the cellsurface membrane (solid arrow) and in the cytoplasm of some ovariancarcinoma cells (A). Immunohistochemical staining of p53 in thenucleus of some ovarian carcinoma cells (B).

(p = 0.5635), all primary lesions (p = 0.4066), and allmetastatic lesions (p = 0.7364).

4. Discussion

The changes in expression of p185 erbB-2 and thenuclear accumulation of p53 have been studied inthe progression of ovarian neoplasia. Several stud-ies have determined differences between benign, bor-derline, and malignant lesions of the ovary with re-spect to nuclear accumulation of p53 and the pheno-typic expression of p185 erbB-2. Anreder et al. [1]observed significantly elevated nuclear accumulationof p53 (p = 0.001) and expression of p185 erbB-2(p = 0.001) in ovarian carcinomas compared to bor-derline tumors. Harlozinska et al. [12] reported no ev-idence of p53 and p185 erbB-2 expression in benignneoplasms of the ovary, 30% p53 and 35% p185 erbB-2

expression in borderline ovarian tumors, and 50% p53and 48.5% p185 erbB-2 expression in ovarian carcino-mas. Similarly, Harlozinska and Bar [11] observed nonuclear accumulation of p53 and expression of p185erbB-2 in benign ovarian tumors and detected carcino-mas, respectively. Mabrouk et al. [14] also observedan overexpression of p185 erbB-2 and mutant p53 pro-teins in malignant cases when compared with benigncases. Overexpression of p185 erbB-2 at the cellularmembrane has been associated with poor survival inovarian carcinogenesis [2,21,24]. Similarly, lack ofnuclear accumulation of p53 or mutations in p53 havebeen associated with significantly greater disease-freesurvival than a high expression of p53 [22].

Most of the studies reporting differences betweenthe expression of p53 and p185 erbB-2 between benignand malignant lesions of the ovary, however, are notcomparisons within the same tumor. Our findings ofno differences in expression of p53 and p185 erbB-2between primary and metachronous metastatic lesionsin patients with advanced chemotherapy resistant ovar-ian carcinoma confirm the findings of a limited num-ber of studies available for p53 and p185 erbB-2 com-parisons within individual tumors. Daidone et al. [5]reported no differences in the nuclear accumulationof p53 between primary and synchronous metastaticsites in 61 women with ovarian carcinomas. Goff etal. [6] observed no significant changes in the nuclearaccumulation of p53 and expression of p185 erbB-2between primary and metachronous metastatic lesionsin 23 women with advanced ovarian cancer. A largerstudy by Tewari et al. [25] also found no significant dif-ferences between the nuclear accumulation of p53 andexpression of p185 erbB-2 in matched cases. Few ofthese studies reporting the expression of p185 erbB-2between primary and metastatic lesions in individualovarian tumors have reported immunostaining of p185erbB-2 by cellular localization. To date, the cellularlocalization of p185 erbB-2 is not fully understood.Nevertheless, we observed immunostaining in the cy-toplasm and in the membrane of tumor cells and did notfind any significant differences between primary andmetastatic lesions based on the cellular localization ofp185 erbB-2.

Our study demonstrates that there are no differ-ences in the nuclear accumulation of p53 and thephenotypic expression of p185 erbB-2 between pri-mary and metastatic lesions (in patients with advancedchemotherapy resistant ovarian carcinoma) for the pur-poses of selecting tumors for novel therapies whichtarget p53 and p185 erbB-2. Thus, when determining


Primary Lesionsp185 erbB-2 Immunohistochemistry Score

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Primary Lesionsp185 erbB-2 Immunohistochemistry Score

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Fig. 2. Immunohistochemistry scores for p185 erbB-2 cytoplasmic (A) and membranous (B) staining among all primary and metastatic lesionsfor the matched cases (n = 26).

appropriate targets, a sample of a primary lesion maybe just as useful as a sample of a metastastic lesion.However, future studies are necessary in determiningif any changes in p53 and p185 erbB-2 are observedbetween early and late stage ovarian carcinomas.

Acknowledgment

Support provided by the Specialized Program of Re-search Excellence (SPORE) in Ovarian Cancer NCI #CA83591-02.


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p53 and erbB-2 are not associated in matched cases of primary …downloads.hindawi.com/journals/dm/2003/108643.pdf · 2019-08-01 · Disease Markers 19 (2003,2004) 11–17 11 IOS