Retinoblastoma and p53 gene product expression in breast carcinoma: Immunohistochemical analysis and clinicopathologic correlation

Retinoblastoma and ~53 Gene Product Expression in Breast Carcinoma: lmmunohistochemical Correlation

Analysis and Clinicopathologic

MICHEL TRUDEL, MD, LOIS MULLIGAN, PHD, WEBSTER CAVENEE, PHD, RICHARD MARGOLESE, JEAN CoTi, MD, AND GILLES GARIEPY, MD We examined

100 breast cancers for retinoblastoma

protein expression by immunohistochemistry

(Rb) and p53

using the PMG3.245

and PAb 1801 antibodies. We assessed percentages of reactive cells and their intensity, as well as staining patterns. The results were correlated with neu protein reactivity and a panel of variables, including age, tumor size and type, nuclear grade, estrogen receptor/ progesterone

receptor

content, and lymph node

status. Retino-

hlastoma protein negativity, either partial or complete, was noted in 47% of cases. Surprisingly,

a relatively stronger Rb reaction was

seen in some high nuclear grade tumors. p53 positivity was found in 23% of cases and was a significant predictor was correlated

with poorly differentiated

plasms and nar expression distribution

profiles

of Rb loss. p53 also

(nuclear grade III) neo-

but not with negative ER status. Tissue

for Rb-negative

and p53-positive

cells were

variable in this series, with both uniform and heterogeneous patterns observed. This suggests that Rb and p53 alterations may represent early or late events in transformation. Our findings further implicate Rb and p53 derangements 23:1388-1394.

Copyright

in mammary oncogenesis.

HUM PATHOL

0 1992 by W.B. Saunders Company

Cytogenetic, molecular, and immunologic studies have uncovered a series of nonrandom genetic changes in breast carcinoma.’ Recurrent karyotypic abnormalities have been found on chromosomes 1, 3, 6, 7, and 11.’ A gene at 17q21 has been linked to inherited susceptibility to breast cancer of early-onset type.” Oncogene aberrations, involving neu (c-erbB2), c-myc, d-2, ru.5, etc,4-” have been detected. Tumor-suppressor genes also have been implicated in mammary neoplasia through studies of familial cancer and analysis of tumor DNAs for loss of heterozygosity. Loss of heterozygosity in breast carcinoma has been noted on five chromosomes at the following loci: 1~34-36,” lq23-32,” 3~21-25,” 11~15,‘” 13q,14 and 17~13.‘~ The latter allelic losses have raised the question of involvement of the retinoblastoma (Rb) and p53 genes. From the Departments of Pathology and Surgery, Sir Mortimer B. Davis:Jewish General Hospital and McGill University, Ludwig Institute for Cancer Research, Montreal, Quebec, Canada; and Department of Pathology, Hi,pital St-Luc and Universitk de Montrial. Montreal, Quebec. Accepted for publication March 24, 1992. Kq words: breast carcinoma, retinoblastoma protein, p53 protein, immunohistologic analysis. Address correspondence and reprint requests to Mic-hel Trudel, MD, Department of Pathology, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste Catherine Rd, Montreal, Quebec, Canada H3T lE2. Copyright 0 1992 by W.B. Saunders Company 004S-8177/92/2312-0012$5.00/O

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MD,

The Rb gene is the prototypic tumor-suppressor gene. This large (200 kb) gene is located at 13q14.‘” It encodes a 1 1 0-kd nuclear protein expressed by normal tissues.17 The protein shows differential phosphorylation during the cell cycle,‘” with the unphosphorylated form predominating in Cl and thought to possess growthsuppressive activity. This may be mediated by binding to transcription factors.‘” Loss-of-function mutations of the Rb gene were first characterized in hereditary and sporadic forms of retinoblastoma.““.” They have subsequently been described in osteosarcomas,‘” soft tissue sarcomas,“’ small cell lung carcinoma,‘” bladder cancer,p5 lymphoid leukemia,“” lymphoma,‘7 etc. In breast neoplasia more specific evidence for Rb gene involvement derives from genomic analysis of breast cancer cell lines, primary tumors, and metastases, which has revealed deletions or alterations in a significant number of cases.2R-3” The p53 gene also has been incriminated in breast malignancy. It is located at 17~13 and spans 20 kb.“’ The protein product is a 53-kd nuclear phosphoprotein whose action remains unclear, although roles in DNA replication and/or transcriptional activation have been postulated.“” p53 has a very short half-life (10 to 20 minutes) and is rapidly degraded, with levels in normal cells being very low. Mutant p53 proteins, on the other hand, are stabilized and may form complexes with wildtype ~53, which recent work has indicated can suppress transformation.“” The role of p53 in neoplasia appears complex, with some tumors showing complete absence of ~53,“~ whereas others display mutations of different types with differing properties.” In some tumors the effect of mutation is loss of suppressive activity, thought to result from neutralization of the wild-type protein by heterodimeric association with the mutant. In other cases the mutant behaves like a bona fide dominant oncogene, eg, conferring a growth advantage when introduced in cells lacking endogenous ~53.“’ The site of mutation and the ratio of mutant to wild-type p53 appear to be critical in determining the net functional outcome. Deletions or mutations of p53 have been identified in colorectal carcinomaJ’~” (particularly in late stages of carcinogenesis), small cell and non-small cell carcinoma of lung,‘i7.JH brain tumors,“” esophageal”” and liver4’ cancer, sarcomas,J4 hematologic neoplasms,“9,4” etc. Indeed, it has been claimed that p53 mutations may

RB AND ~53 EXPRESSION

IN BREAST CARCINOMA

represent the most frequent genetic abnormality in human cancer.‘” 1’53 abnormalities also have been linked to breast carcinoma through molecular analyses demonstrating loss of heterozygosity at 17~13. 144 as well as specific somatic’” or germline4” mutations. In addition, immunologic st.udies have shown p53 accumulation in breast tumor tissue, as well as raised levels of anti-p53 antibodies in the sera of breast cancer patients.47,4” To confirm and extend these observations we undertook thse present study to verify the incidence and pattern of Rb and p53 protein expression in a series of 100 breast carcinomas, and to correlate the results with a broad panel of clinicopathologic variables, including age, tumor size, histotype, nuclear grade, presence and number of positive axillary nodes, and estrogen receptor/progesterone receptor content. Immunohistochemistry was used to increase the chance of detecting abnormalities that might otherwise remain occult by hybridization techniques. We also wished to examine Rb and p53 protein expression in conjunction with each other and with the neu protein, to determine whether there are coordinate alterations in expression, and to assess whether such sets of alterations (if any) define specific suhzsets of tumors. Finally, we attempted to relate the profiles of immunoreactivity to the staining patterns that would he expected if the genetic changes accompanied tumor initiation or progression. MATERIAL!5

AND METHODS

Sample Preparation Fw4i specimens of 100 primary breast tumors were obtained intraoperatively. embedded in cryopreservative (OCT c~q~ound), snap-frozen in cooled isopentane, and stored at - /O”(:. For~r to six micrometer-thick cryostat sections were fixed fi~r IO minutes in cold acetone and were briefly air-dried. ‘Tumor typing and nuclear grading were performed on hem~~toxylin-eclsirl-stained sections of formalin-fixed. paraffinembedded ttssuc in each case. I,evels of estrogen and progesterone receptor-s were determined by a standard biochemical assay. lmmunocytochemistry (:r‘yc)stat sections were preincubated in 3% H,Op for 10 minutes, followed by 10% nonimmune blocking serum for 10 minutes. The following monoclonal antibodies were then applied separately to serial cryostat sections and incubated for I ltiour at room trmperature in a humidified chamber: PMG3245.1 I (Ig(;, isotype; PharMingen, San Diego, CA; 1:50 diIulion in ‘Iris), which recognizes the 1 10-kd Rb gene product; PAb 180 1 (p53 Ah-2; Oncogene Science, Manhasset, NY; 1: 50 dilution in Tris). which recognizes a human-specific epitope near the amino terminus of the ~53 protein and detects both normal and mutant forms of ~53; and 3B5 (Oncogene Science C-UPUAb-3; 1:25 dilution in Tris), directed against a peptide sequence frl,m the carboxyl domain of the ECUgene product. Subsequently, the slides were exposed to biotinylated rabbit anti-mouse secondary antibody for 30 minutes, followed by streptavitiin-peroxidase conjugate for 30 minutes. Washes in ‘fris buflt*l- were performed between each of the preceding steps. Finally, diaminobenzidine substrate solution was applied. .I‘cIfacilitate both detection of positive cells and determination of the per-ccntages thev represented, consecutive slides were

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(Trudel et al)

processed with and without a light hematoxvlin c.ounterstain and compared. The following were used as controls: Retinobla~torna: The Rb-positive U-20s osteosartoma cell line served as a positive control. Staining of normal breast epithelium, when present in the section, also provided a positive internal control. The Rb-negative WERI-27 Rb cell line was used as a negative control. Additional negative controls were produced by substitution of an irrelevant mouse IgG, monoclonal antibody for the primary antibody or omission of the latter. p5?: Sections of colonic and breast carcinomas with established high p53 expression were used as positive controls. Substitution of a nonspecific mouse monoclonal antibody for the primary antibody and omission of the latter served as negative controls. neu: A breast carcinoma with strong neu expression served as a positive control. The specificity of the reaction was verified by abrogation of. staining following preincubation with a blocking nzpu peptide (Oncogene Science).

Quantitation

and Analysis

Semiquantitative estimation of reactivity for Rb and ~53 was performed by assessing approximate perctantages of positive cells. Patterns of staining, such as uniform versus clusters or scattered cells, also were noted. Only definite staining was scored as positive; faint or equivocal staining was considered negative for interpretive purposes. Expression of neu was graded from 0 to 2 according to intensity (0, absent or equivocal; 1, present; 2, strong). Slides were scored by two independent observers. Reproducibility of scoring also was assessed by re-examining 30 cases on two separate occasions and proved acceptable. Evaluation was carried out without knowledge of the clinical and laboratory information derived from the patients’ records. The follow-up period in virtually all cases was too brief (6 to 18 months) to allow meaningful survival analysis at this time. Data were entered on a Macintosh SE-30 (Apple, Capertint), (:A) and statistical analysis was carried out with Statistical Package for the Social Sciences (SPSS Inc, Chicago, II.) software.

RESULTS The descriptors in Table 1.

of the case population

are indicated

Reaction Patterns Relznoblartomu. Retinoblastoma protein was localized to the nucleus. The level of Rb expression showed considerable variation in this series. Over half of the cases (53%) displayed Rb positivity in essentially all tumor cells, with only minor variation in staining intensity. In this category an intense positive reaction was noted in several grade 3 tumors (Fig 1). and this reaction appeared more pronounced than in normal breast epithelium. Twenty-eight percent of cases showed heterogeneous Rb reactivity, with variable numbers of negative cells (Fig 2). Unreactive cells formed clusters of variable size or were scattered throughout the neoplasm. Negative cells were occasionally identified in the in situ component of a carcinoma. Nineteen percent of specimens showed no visible reaction fw Rb protein. Thus, 47% of cases demonstrated some degree of Rb negativity, either partial or complete.

HUMAN PATHOLOGY

TABLE 1.

Studv Potxlation

Total no. of cases Sex Mean age, yr (range) Mean (SD) and median diameter (range) Histologic type Ductal (?DCIS) Lobular Mutinous Medullary Other Nuclear grade I (favorable) II (intermediate) III (poor) Stage* I II 1-4 positive nodes 5- 10 positive nodes > 10 positive nodes ER Evaluable cases Median value (fmol/mg) Positive cases (> 10 fmol/mg) PR Evaluable cases Median value (fmol/mg) Positive cases (> 10 fmol/mg)

Volume 23, No. 12 (December

Characteristics 100 98 F; 2M 56 (28-84) 2.6 cm (1.86). 2.0 cm (0.5-10) 85 4 3 3

64 31 50 38 27 8 3 90

39 61 81 60 55

* Staging information not available in 12 cases. Abbreviations: DCIS, ductal carcinoma in situ; ER, estrogen receptors; PR. progesterone receptors.

~53. p53 staining was confined to neoplastic cells, with no reaction in normal ductal or lobular epithelium. p53 positivity also was restricted essentially to the nucleus, with only rare cells displaying faint cytoplasmic reactivity. A positive reaction was noted in approximately one fourth of cases (23%). In 18 of the positive cases, there was moderate to strong staining throughout the tumor (Fig 2); scattered clusters of positive cells were noted in the remainder. Two of three p53-positive invasive carcinomas that contained an evaluable in situ component also showed staining in that component. Statistical

Correlations

Retinoblastoma and p53 expression were subjected to bivariate and multivariate analysis using multiple linear regression. Presence of p53 and degree of positivity were significantly associated (P = .Ol) with increasing nuclear grade on bivariate analysis. The correlation between a positive p53 reaction and high nuclear grade was strengthened on logistic regression (P = .0064). An association with neu expression (Fig 2) also was evident (P = .02). Positivity was equally distributed among stage I and II cases. Presence of p53 and an increasingly strong reaction also were correlated with partial and complete loss of Rb reactivity (P = .0021 and P = .0167, respectively). Interestingly, Rb positivity showed a weakly significant (P = .045) association with grade 3 tumors. There was no discernible correlation of Rb or p53 with the other parameters examined: age, tumor size, histotype, level 1390

1992)

of estrogen receptor or progesterone receptor, presence and number of positive axillary nodes.

and

DISCUSSION In this study we examined the expression of the Rb and p53 proteins, as well as their relationship to each other and to standard prognostic indicators, in a series of 100 primary human breast cancers. This follows several recent observations that have suggested a role for these genes in mammary oncogenesis. Retinoblastoma gene inactivation has been noted in two of nine breast cancer cell lines by Lee et a1.28 T’Ang et al identified homozygous internal deletions and total deletions of the Rb gene, with absent or truncated Rb transcripts, in four of 16 cell lines and in three of 41 primary tumors.*’ Varley et al showed structural abnormalities of the Rb gene in 15 of 77 primary carcinomas.30 When studied by immunohistochemistry, 16 of 56 cases (29%) in their series contained tumor cells negative for Rb protein. All tumors displaying a genetic alteration to Rb, with one exception, had a proportion of negative cells by immunostaining. In the present series altered expression of the Rb gene product was seen more frequently. Heterogeneous reactivity was noted in 28% of cases and complete absence of reaction was noted in 19% of cases. It should be noted that the anti-Rb monoclonal antibody used in the study of Varley et a13’ (RBlAb20) is different from the antibody used here, and variable availability of the respective target epitopes may account for the different results. It also is conceivable that some of the apparently nonreactive cells contain levels of functional protein below the threshold for detection by this immunocytochemical technique. Enhanced Rb positivity was incidentally noted in some grade III neoplasms. There is clearly an element of subjectivity in this type of assessment; however, statistical analysis did support this association. Such “overexpression” seems paradoxical and its cause is unclear, although the possibility of a greater amount or more stable form of Rb protein in some poorly differentiated cells is suggested. There also is substantial evidence for p53 involvement in breast carcinoma. In a large series of breast cancer patients Coles et al have detected loss of heterozygosity at 17~13.1, which includes the p53 gene, in 27% to 47% of cases.44 Nigro et al have uncovered p53 mutations in two cell lines and one primary tumor.45 Davidoff and colleagues have documented p53 alterations maintained throughout progressive stages of breast cancer.4g.50 In addition to these somatic changes, germline p53 mutations have been demonstrated4’j in the LiFraumeni syndrome, a familial cancer syndrome of diverse tumors, the most frequent of which (25%) is breast cancer. Twenty-three percent of specimens in our series showed p53 immunoreactivity. This is comparable to the results of Davidoff el a15’ (11 of 49 breast cancers or 22%), but somewhat less than reported by others. Cattoretti et a1,5’ in the portion of their study using the PAb 1801 antibody, detected p53 positivity in 45% (40

RB AND

~53 EXPRESSION

IN BREAST CARCINOMA

(Trudel et al)

FIGURE 1. (lop) Invasive ductal carcinoma of breast, nuclear grade Ill (hematoxylin-eosin stain; magnification x400) showing (bottom) strong expression of Rb protein in most cells (immunoperoxidase without counterstain; magnification X400).

of 8X cases) and Ostrowski et al”” detected positivity in 35% (32 of 90 cases). Potential explanations for the variability of these results include differences in immunohistotrhernical technique and differing definitions of a positive reaction (eg, in any cell u the majority of cells). It is apparent that p53 reactivity is seen in a significant minority of mammary carcinomas. Our analysis corroborates a strong association between p53 expression and poor nuclear grade rumors, as noted by others.“‘.” An association with ~LPUamplitication or overexpression”” also is sustained. In contrast to Cattoretti et al, who showed a significant correlation 1391

with increased proliferative activity (high Ki-6’7 scores) and negative estrogen receptor status on bivariate analysis does not indicate an study,” our multivariate independent correlation with negative estrogen receptor status. The emerging evidence from these and other”” studies, however, does suggest that 1~53 xs a marker of more poorly ditierentiated carcinomas, possihly with more aggressive behavior. Whether ~53 txpression will carry predictive power independent of more traditional variables, such as tumor size and lymph node status, awaits accumulation of large series with adequate fitllow-up.

FIGURE 2. (Top left) Invasive ductal carcinoma, nuclear grade Ill (hematoxylin-eosin stain; magnification x400) showing (top right) subtotal loss of Rb expression with scattered residual positive cells (arrowheads), (bottom left) moderate to strong reaction for ~53 in majority of cells, and (bottom right) strong neu positivity (all immunoperoxidase without counterstain; magnification X400).

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RB AND ~53 EXPRESSION

IN BREAST

An important issue concerns the point in tumor evolution at which a gene is implicated, and it has been prcjposed’” that the immunostaining patterns for Rb and ~53 may reflect this. For instance, if no Rb-positive cells are present (or if there is homogeneous ~53 expression in all cells)., this would suggest that the gene is related to neoplastic initiation. On the other hand, if reactivity is heterogeneous with a mixture of positive and negative cells (or alterations confined to the metastasis), a progrtlssional role would be more likely. Both patterns of expression were encountered in this series. The identification of‘complete Rb negativity in 19% of our cases. together with the finding of uniform ~53 expression in 18% of carcinomas as well as in foci of ductal carcinoma in situ, is in keeping with alterations occurring early in the cascade of neoplastic transformation. In the study also were found in of Davidoff et ~1,~” 1’53 alterations foci of carcinoma in situ and were maintained throughout subsequent stages of infiltrating and metastatic carcinoma. Hmowever. staining also was heteromorphic in other cases in our series. This would favor a progressional function for these changes, as is more generall) suggested by the diversity of tumor types in which mutations of these genes, particularly p5Y, have been incriminated and their frequency of occurrence. The situation may be analogous to that in colorectal cancer in which, as noted by Vogelstein et al,“” the time of occurrence of’ mutations varies between different tumors and may be less important for oncogenesis than the accretion of a11 mutational events. The finding that p5:3 expression co-exists with, and is a significant predictor of, Rb loss raises the interesting question of’ some form of collaboration. Similar observations of c.oincident alterations in Rb and p53 have been made in some soft tissue sarcomas”” and leukemic T-cell lines.‘” The situation in these nonvirally induced nt~oplasms echoes that of several DNA tumor viruses, such as SV40, human papillomavirus types 16 and 18, and some adenoviruses. The oncoproteins produced b) these viruses are now known to bind Rb and p53 gene products s’eparately, thereby abrogating their effects.‘7 In aggregate, these findings suggest that these genes ccmtrol distinct 01. redundant regulatory pathways in the cell cycle, and that in various neoplasms. inclucling some brea:jt cancers. both pathways are inactivated, either through mutation or complexing with viral OIICOproteins, 2~spart of the neoplastic process. These regulatory c.irc.uits are und(tubtedly modulated at the gene and/or protein levels bv the integration of various microenvirollmrrntal and intracellular signals. The tissue specificity of these molecular interactions may in turli influence I:he distribution of neoplasms that otherwise share abnormalities in the Rb and/or ~53 genes. Ehcidation of these regulatory networks will help clarify the relevant cmtributions of Rb and ~53 alterations to

mammary

carcinogenesis.

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CARCINOMA

(Trudel et al)

HUMAN PATHOLOGY

Volume ‘23, No. 12 (December

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