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p53 Mutation as a Genetic Trait of Typical Medullary Breast Carcinoma

Patricia de Cremoux, Anne Vincent Salomon, Stephane Liva, Rémi Dendale, Brigitte Bouchind'homme, Emmanuel Martin, Xavier Sastre-Garau, Henri Magdelenat, Alain Fourquet, Thierry Soussi

Affiliations of authors: P. de Cremoux (Department of Pathology and Universite Paris 7), A. V. Salomon, B. Bouchind'homme, X. Sastre-Garau (Department of Pathology), S. Liva, E. Martin (Department of Translational Research), R. Dendale, A. Fourquet (Department of Radiotherapy), H. Magdelenat (Departments of Pathology and Translational Research), T. Soussi (Unité Mixte de Recherche 218, Centre National de la Recherche Scientifique), Institut Curie, Paris, France.

Correspondence to: Patricia de Cremoux, M.D., Ph.D., Department of Pathology, Institut Curie, 26 rue d'Ulm, 75248, Paris cedex 05, France (e-mail: Patricia.De-Cremoux{at}curie.fr).

Medullary carcinoma is a poorly differentiated breast cancer tumor with a high histologic grade and a paradoxically good prognosis. It accounts for only 5% of all breast cancers (1-3). Thus far, only histologic criteria are used to define this tumor type; no genetic characteristics have been identified. An alteration in the p53 gene (also known as TP53) is found in 20%-40% of invasive breast cancers, but its status in medullary breast cancer is poorly documented. Immunochemical detection of a stable mutant p53 in nuclei of tumor cells is the most convenient assay, but the correlation of the results from such an assay with results from molecular analysis, such as sequencing, is not totally in agreement (4). The frequency of p53 mutations in breast cancer is around 20%, but immunohistochemical analysis detects p53 accumulation in 30%-40% of tumors (5,6).

In the present study, we demonstrate that typical medullary breast carcinomas have a high frequency of p53 alterations (100%). We found excellent agreement between the results of the different analytical techniques that we used.

We obtained specimens from 23 medullary breast carcinomas from the Institut Curie tumor bank; these carcinomas were initially registered (during the period from 1989 to 1995) as medullary carcinomas. All specimens were selected on the basis of the availability of blocks and frozen material, and two trained pathologists (B. Bouchind'homme and A. V. Salomon), who were blinded to the status of the specimens, independently reviewed each specimen during the study. As defined by Rapin et al. (3), typical medullary carcinomas display at least 75% syncytial architecture, a well-defined border, diffuse lymphoplasmocytic infiltrate, moderate or marked anisonucleosis, and absence of tubular differentiation and/or an intraductal component (2,3) (15 of our 23 specimens). Atypical medullary carcinomas (eight of our 23 specimens) have a syncytial architecture and two or three of the above criteria.

We assessed the status of the p53 gene by using molecular and immunohistochemical techniques (8) (Fig. 1).Go For the molecular analysis, we combined direct sequencing of genetic material from tumors with a functional assay, developed by Flaman et al. (7), to ensure that all molecular changes detected were deleterious for p53 function (true mutations). Seventy-four percent (17 of 23) of tumors had a p53 mutation (Table 1).Go Immunohistochemical analysis showed that p53 accumulated in the 14 tumors that contained a missense mutation, one in-frame codon deletion (patient 7), or a long truncated protein (patient 3) (Table 1Go). Tumors expressing short truncated p53 (tumors in patients 2, 10, and 15) did not accumulate p53, since such proteins are rather unstable (Table 1Go).



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Fig. 1. Histology and p53 immunostaining of medullary breast cancer. Sections (4 µm thick) were cut from formalin-fixed, paraffin-embedded tissue. p53 protein was stained with DO7 antibody (Dako Corp., Carpinteria, CA). Staining was performed by use of the streptavidin-biotin-peroxidase complexes and diaminobenzidine (Dako Corp.) as chromogen as previously described (9). In A and B, two consecutive histologic sections from a typical medullary carcinoma were stained with hematoxylin-eosin or with a p53 antibody (DO7), respectively. In C and D, two consecutive histologic sections of an atypical medullary carcinoma were stained with hematoxylin-eosin or with a p53 antibody (DO7), respectively. The syncytial architecture and mononuclear infiltrate are present in both types of medullary carcinomas, but glandular structures are never observed in typical medullary carcinomas. Scale bars = 10 µm. Nuclear immunostaining of the p53 protein was scored as the percentage of positive cells. Panels E-H: For the functional and molecular analysis of p53 status, total RNA was purified from frozen tumor material, and complementary DNA (cDNA) was made from this RNA by reverse transcription and amplification by polymerase chain reaction (PCR). Purified PCR products were sequenced bidirectionally by use of a DyeDeoxy Terminator kit (The Perkin-Elmer Corp., Foster City, CA) and an automated sequencer (ABI Prism 310 Genetic analyzer; The Perkin-Elmer Corp.). For all patients, p53 cDNA was sequenced from codon 41 (exon 3) to codon 365 (exon 10). For all but one patient, we studied the transcriptional activity of the p53 protein by using the yeast functional assay previously described by Flaman et al. (7). Transcriptional activation is the critical biochemical function of p53, which underlies its tumor suppressor activity. Mutant p53 proteins fail to activate transcription. Furthermore, such transcriptional activity is functional in yeast, and p53 mutants that are inactive in humans are also inactive in yeast. The recipient yeast strain (yIG397) is defective in adenine synthesis because of a mutation in its endogenous ADE2 gene, but it contains a second copy of the ADE2 open reading frame controlled by a p53 response promoter. Because ADE2 mutant strains grown on low adenine plates turn red, yIG397 colonies containing mutant p53 are red, whereas colonies containing wild-type p53 are white. Yeast strain yIG397 was cotransformed with PCR-amplified p53 and a linearized expression vector (pSS16), and the p53 cDNA was cloned in vivo by homologous recombination. We visually quantified the percentage of red colonies and of white colonies in each assay. As previously shown by Flaman et al. (7), more than 10% of red colonies is always associated with a p53 mutation. We sequenced pooled plasmids from red colonies. In all cases, the mutation matched mutations found by direct sequencing of the tumor cDNA. To minimize mutations introduced during PCR, we have used Pfu DNA polymerase (Stratagene Cloning Systems, La Jolla, CA), a high-fidelity polymerase. Yeast culturing and transformation were done as described by Flaman et al. (7). In E and F, yeast assay from tumors from patients 5 and 1 shows 8% and 85% red colonies, respectively. In G and H, p53 sequences are shown from tumor from patient 5 (wild type) and tumor from patient 21 (mutant) at position 205.

 

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Table 1. Histologic, molecular, and immunochemical status of medullary breast carcinoma

 
Thus, we observed 100% agreement between the molecular genomic analysis and the accumulation of p53 in the nucleus of the tumor cells. In this immunohistochemical analysis, all positive cases were intensely stained, with more than 70% of cells staining (Fig. 1Go). This result is very unusual for invasive breast cancer, because the staining intensity is generally quite heterogeneous. This finding is not due to technical variation, because we observed a similar heterogeneity in 328 breast tumor samples that we have analyzed recently (9). Previous studies (10-12) have suggested that the p53 alteration could occur frequently in medullary breast carcinoma, but no detailed analyses combining sequencing and immunohistochemistry have been performed; furthermore, the small number of cases reported did not enable adequate statistical evaluation.

As defined by Rapin et al. (3), eight tumors were atypical medullary breast carcinomas and 15 tumors were typical medullary breast carcinomas. It is interesting that all typical medullary carcinomas harbored a p53 alteration (Table 1Go) (95% confidence interval [CI] = 78%-100%). In the eight atypical medullary carcinomas examined, only two mutations were detected, giving a frequency of 25% (95% CI = 3%-65%), a frequency similar to that in invasive breast carcinomas.

Our results indicate that medullary breast carcinoma is a distinct genetic subtype of breast cancer. The observations of a high incidence of p53 alterations and an unambiguous pattern of p53 staining suggest that the genetic pathway leading to this subtype of cancer is different from that of common infiltrating breast carcinoma. To our knowledge, this is the first time that a genetic defect is able to specifically distinguish medullary breast carcinoma. It has recently been shown (13,14) that medullary breast carcinoma is found at a very high frequency (up to 20%) in patients with BRCA1-associated breast cancers. The observation that BRCA1-associated tumors also have a high frequency of p53 mutation (15) suggests that there may be a relationship between alterations in the BRCA1 gene and medullary breast carcinoma, supporting the notion that a specific genetic pathway leads to this tumor type.

Medullary breast carcinomas usually have a much more favorable prognosis than the poorly differentiated common invasive cancers. This behavior is paradoxical compared with the behavior of sporadic invasive breast cancer but not with the behavior of BRCA1-associated tumors. It has been proposed (16,17) that substantial heterogeneity exists in the behavior of the various p53 mutants toward both the biologic activity of p53 and their specific association with some cancer types or various clinical parameters. In our study, we did not find any distinctive particularities in the various p53 mutations. The fact that tumor cells harbor a multitude of genetic defects usually associated with a given type of cancer suggests that survival or response to therapy is not associated with a single genetic defect but may result from a combination of various defects. The present data indicate that p53 alteration is an important factor in typical medullary carcinoma but probably combines with other known or unknown specific genetic defects in order to explain the paradoxically increased sensitivity to treatment of this subtype of tumor. The fact that p53 and BRCA1 are both associated with DNA repair is a strong argument in favor of such a hypothesis.

Finally, if confirmed by independent studies, our results indicate that p53 status is an important component in typical medullary breast carcinoma, and a determination of the status of p53 in the tumor should be included in its diagnosis.

NOTES

Supported by grants from the Ligue Nationale contre le Cancer (Comité de Paris and Comité Nationale), the Association pour la Recherche contre le Cancer, Mutuelle Générale de l'Éducation Nationale, Fondation pour la Recherche Médicale, Fédération des Groupements d'Entreprises dans la Lutte contre le Cancer, and Commission de Recherche Clinique of Institut Curie.

We thank Dr. G. Contesso and Dr. J. Bram for their critical reading of the manuscript, J. P. Laborde for iconography, and C. Rouillon for technical assistance.

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Manuscript received September 28, 1998; revised January 22, 1999; accepted February 1, 1999.


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