Benign Breast Tissue Samples Research Articles

Expression profiles of small non-coding RNAs in breast cancer tumors characterize clinicopathological features and show prognostic and predictive potential

Precision medicine approaches are required for more effective therapies for cancer. As small non-coding RNAs (sncRNAs) have recently been suggested as intriguing candidates for cancer biomarkers and have shown potential also as novel therapeutic targets, we aimed at profiling the non-miRNA sncRNAs in a large sample set to evaluate their role in invasive breast cancer (BC). We used small RNA sequencing and 195 fresh-frozen invasive BC and 22 benign breast tissue samples to identify significant associations of small nucleolar RNAs, small nuclear RNAs, and miscellaneous RNAs with the clinicopathological features and patient outcome of BC. Ninety-six and five sncRNAs significantly distinguished (Padj < 0.01) invasive local BC from benign breast tissue and metastasized BC from invasive local BC, respectively. Furthermore, 69 sncRNAs significantly associated (Padj < 0.01) with the tumor grade, hormone receptor status, subtype, and/or tumor histology. Additionally, 42 sncRNAs were observed as candidates for prognostic markers and 29 for predictive markers for radiotherapy and/or tamoxifen response (P < 0.05). We discovered the clinical relevance of sncRNAs from each studied RNA type. By introducing new sncRNA biomarker candidates for invasive BC and validating the potential of previously described ones, we have guided the way for further research that is warranted for providing novel insights into BC biology.

Application of a Deep-Learning Technique to Non-Linear Images From Human Tissue Biopsies for Shedding New Light on Breast Cancer Diagnosis.

The development of label-free non-destructive techniques to be used as diagnostic tools in cancer research is of great importance for improving the quality of life for millions of patients. Previous studies have demonstrated that Third Harmonic Generation (THG) imaging could differentiate malignant from benign unlabeled human breast biopsies and distinguish the different grades of cancer. Towards the application of such technologies to clinic, in the present report, a deep learning technique was applied to THG images recorded from breast cancer tissues of grades 0, I, II, and III. By the implementation of a convolutional neural network (CNN) model, the differentiation of malignant from benign breast tissue samples and the discrimination of the different grades of cancer in a fast and accurate way were achieved. The obtained results provide a step ahead towards the use of optical diagnostic tools in conjunction with the CNN image classifier for the reliable and rapid malignancy diagnosis in clinic.

Abstract 3492: Subtype-specific expression of small non-coding RNAs in breast cancer

Abstract We have performed a small RNA-sequencing (RNA-Seq) experiment to find candidate small non-coding RNAs (ncRNAs) in breast cancer (BC) for the identification of signatures that aid in the prediction of therapy responsiveness, prognosis or patient outcome. We used 195 invasive BC tumor and 20 benign breast tissue samples from the Kuopio Breast Cancer Project (KBCP) study material. KBCP material is collected from a genetically homogeneous population of Eastern Finland, and includes fresh-frozen and FFPE breast tissue samples, EDTA blood samples and serum, as well as comprehensive background/lifestyle information, clinical, treatment and follow-up data extending over 20 years. This well-defined material enables the identification of factors associating with BC risk, different stages of disease progression and outcome. Total RNA was extracted from the fresh-frozen BC and benign breast tissue using the Ambion mirVana miRNA Isolation Kit. Small RNA-Seq was performed using the Illumina TruSeq Small RNA Library Prep kit and the Illumina MiSeq next-generation sequencing instrument. Analyses were concentrated in PIWI-interacting RNAs (piRNAs) and other non-miRNA small ncRNAs (sncRNAs). Bioinformatic analysis commenced with quality control and preprocessing steps including read quality assessment (FastQC), adapter trimming (TRIMMOMATIC), and removal of e.g. ribosomal RNA reads (Bowtie). Preprocessed reads were aligned (Tophat) to human reference transcriptomes (piRNABank and GENCODE non-miRNA sncRNAs), followed by data conversions e.g. for visualization (samtools, IGVtools). Gene-wise read counts were collected using summarizeOverlaps R package, and statistically differentially expressed (DE) genes were identified using DESeq2 R package. We first compared the small RNA expression in the benign breast tissue with invasive BC and observed in total 635 sncRNAs and 94 piRNAs with differential expression (PAdj&amp;lt;0.05). Of these 520 sncRNAs and 73 piRNAs were upregulated, and 115 sncRNAs and 21 piRNAs downregulated in the invasive tumors compared to benign breast tissue. When comparing the luminal BC subtype with triple-negative BC (TNBC) we observed 204 DE (PAdj&amp;lt;0.05) sncRNAs and 32 piRNAs. Of these, 103 sncRNAs and 18 piRNAs were upregulated, and 101 sncRNAs and 14 piRNAs downregulated in TNBC compared to luminal tumors. Differential expression was also observed between ER (estrogen receptor) negative and ER positive invasive tumors. Of the 242 DE sncRNAs and 32 piRNAs, 105 sncRNAs and 12 piRNAs were upregulated, and 137 sncRNAs and 20 piRNAs downregulated in ER positive BC compared to ER negative. Preliminary results of hierarchical clustering and principal component analysis of normalized log2 piRNA and sncRNA expression levels revealed the potential for finding differences between BC subtypes. The subsequent statistical analysis enabled us to identify individual piRNAs and other sncRNAs as candidate druggable biomarkers for e.g. TNBC patients. Citation Format: Jaana M. Hartikainen, Sami Heikkinen, Maria Tengström, Veli-Matti Kosma, Arto Mannermaa. Subtype-specific expression of small non-coding RNAs in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2017-3492

PLA2G7 associates with hormone receptor negativity in clinical breast cancer samples and regulates epithelial-mesenchymal transition in cultured breast cancer cells.

Breast cancer is the leading cause of cancer‐related deaths in women due to distinct cancer subtypes associated with early recurrence and aggressive metastatic progression. High lipoprotein‐associated phospholipase A2 (PLA2G7) expression has previously been associated with aggressive disease and metastasis in prostate cancer. Here, we explore the expression pattern and functional role of PLA2G7 in breast cancer. First, a bioinformatic analysis of genome‐wide gene expression data from 970 breast samples was carried out to evaluate the expression pattern of PLA2G7 mRNA in breast cancer. Second, the expression profile of PLA2G7 was studied in 1042 breast cancer samples including 89 matched lymph node metastasis samples using immunohistochemistry. Third, the effect of PLA2G7 silencing on genome‐wide gene expression profile was studied and validated in cultured breast cancer cells expressing PLA2G7 at high level. Last, the expression pattern of PLA2G7 mRNA was investigated in 24 nonmalignant tissue samples and 65 primary and 7 metastatic tumour samples derived from various organs using qRT‐PCR. The results from clinical breast cancer samples indicated that PLA2G7 is overexpressed in a subset of breast cancer samples compared to its expression in benign breast tissue samples and that high PLA2G7 expression associated with hormone receptor negativity as well as with poor prognosis in a subset of breast cancer samples. In vitro functional studies highlighted the putative role of PLA2G7 in the regulation of epithelial‐mesenchymal transition (EMT)‐related signalling pathways, vimentin and E‐cadherin protein expression as well as cell migration in cultured breast cancer cells. Furthermore, supporting the findings in breast and prostate cancer, high PLA2G7 mRNA expression was associated with metastatic cancer in four additional organs of origin. In conclusion, our results indicate that PLA2G7 is highly expressed in a subset of metastatic and aggressive breast cancers and in metastatic samples of various tissues of origin and promotes EMT and migration in cultured breast cancer cells.

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Abstract P6-06-02: Low expression levels of hormone receptors (ER-α and PGR) in human breast cancer samples is significantly associated with high-grade human cytomegalovirus-IEA

Abstract P6-06-01: High-grade human cytomegalovirus IEA is associated with expression of COX-2 and 5-LO in human breast cancer samples

Abstract Background: The role of cyclooxygenase-2 (COX-2) in breast cancer development and progression has been supported by an increasing number of studies that show the overexpression of COX-2 in all the stages of the disease but in particular, in the metastatic phase. Besides COX-2 and its derived metabolites, 5-lipoxygenase (5-LO) and leukotrienes have also been associated with cancer progression. Human Cytomegalovirus (HCMV) detection in samples from primary BC, sentinel lymph nodes and brain metastases obtained from breast cancer patients' suggests that viral infection may also have a critical role in the development of breast cancer metastasis. Interestingly, in vitro studies showed that HCMV infections induce COX-2 in human fibroblasts, which augments viral replication through a prostaglandin dependent pathway. Thus, our main objective was to investigate whether there is a correlation between HCMV infection and overexpression of COX-2 and 5-LO in breast cancer. If so, HCMV could be an important additional target for breast cancer treatment. Material and Methods: Paraffin embedded breast cancer biopsies (n=48), ductal carcinoma in-situ (DCIS, n=14) and adjacent, benign breast tissue samples (n=29) were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA) proteins, COX2 and 5LO by using immunohistochemical techniques established in our laboratory. Clinical data were available from the patients´ hospital files provided from the departments of oncology and pathology at Akershus University Hospital, Norway. All patients underwent direct surgery in 2011. All patients received standard adjuvant treatment according to the Norwegian guidelines. For in vitro studies, breast cancer cell lines (MCF-7, MB-MDA-231 and SKBR3) were infected with HCMV VR1814 strain and levels of COX-2 and 5-LO were determined by qPCR and western blot and immunofluorescence. Results: High levels of COX-2, 5-LO and HCMV-IE were detected mainly in breast cancer samples. High grade HCMV-IE (defined as &amp;gt;50% positive cells in the tumor tissues) was detected in 72% of infiltrating BC and in 28% of DCIS, but it was detected only in 7% of benign, adjacent breast tissue samples. Similarly, high grade COX-2 and 5-LO were detected in 58% and 53% of BC, in 21% and 8% of DCIS, and in 4% and 7% of benign, adjacent breast tissue samples, respectively. We found a statistically significant positive correlation for the levels of HCMV-IE and COX-2 (p=0.001) as well as for HCMV-IE and5-LO (p=0.0002) in infiltrating breast cancer. Furthermore, induction of COX-2 and 5-LO was confirmed in breast cancer cell lines following infection with HCMV was shown at both mRNA and protein level. Conclusion: Our findings confirm a positive correlation of HCMV-IE protein synthesis and overexpression of COX-2 and 5-LO in infiltrating breast cancer, DCIS and benign, adjacent breast tissue samples, which is consistent with the up-regulation of these enzymes in breast cancer cells infected with HCMV. These results suggest that the inflammation driven by COX-2 and 5-LO in human breast cancer might be induced by HCMV infections and lead to tumor progression. Thus, anti-viral therapy should be considered as an additional experimental treatment in selected breast cancer patients. Citation Format: Costa H, Touma J, Davoudi B, Geisler J, Vetvik K, Söderberg Naucler C, Rahbar A. High-grade human cytomegalovirus IEA is associated with expression of COX-2 and 5-LO in human breast cancer samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-06-01.

The relationship between Human Papillomavirus and Epstein-Barr virus infections with breast cancer of Iranian patients

Background and Aim: As the etiology and progression of breast cancer r emain incompletely understood, novel routes of disease pa thogenesis are important to consider. Some researchers have been found Human papillomavirus (HPV) and Epstein Barr virus (EBV) in breast carcinomas (BCs). Methods: Paraffin-embedded sections from 65 female patients with breast carcinoma and 53 breast tissues from patients with fibrocystic disease as control were selected. After DNA extraction and amplificati on of housekeeping gene (beta-globin), all suitable samples were evaluated for presence of DNA-EBV and DNA-HPV by using PCR (Polymerase Chain Reaction). Results: HPV DNA sequences were detected in 3 of the 53 benign breast tissue samples but none of the breast carcinoma samples was identified. EBV was detected by PCR in 23 of 65 (35.38%) cases of breas t cancer specimens and 11 of 53 (20.75%) control samples (fibroadenoma 7 of 26 (26.92%) and fibrocystic 4 of 27 (14.81%)) . A total of 118 samples from 34 cases (81/28%) were positive. Statistically, crammer indicator analysis for EBV i nfection in tumor samples and normal samples was 0.46 which indicates a significa nt relationship. Conclusion: Our analysis could not confirm a role of HPV in br east cancer but statistically, significant correlation between EBV infection and breast cancer exists. To demonstrate the possible relationship be tween viral load and breast cancer, need for epidemiological, biological and mo lecular mechanisms to clear the virus is involved in the process of carcinogene sis.

X-ray scattering for the characterization of lyophilized breast tissue samples

This work investigates the possibility of characterizing breast cancer by measuring the X-ray scattering profiles of lyophilized excised breast tissue samples. Since X-ray scattering from water-rich tissue is dominated by scattering from water, the removal of water by lyophilization would enhance the characterization process. In the present study, X-ray scattering profiles of 22 normal, 22 malignant and 10 benign breast tissue samples are measured. The cut-offs of scatter diagrams, sensitivity, specificity and diagnostic accuracy of three characterization parameters (full width at half maximum (FWHM) for the peak at 1.1nm−1, area under curve (AUC), and ratio of 1st to 2nd scattering peak intensities (I1/I2%)) are calculated and compared to the data from non-lyophilized samples. Results show increased sensitivity (up to 100%) of the present data on lyophilized breast tissue samples compared to previously reported data for non-lyophilized samples while the specificity (up to 95.4%), diagnostic accuracy (up to 95.4%) and receiver operating characteristic (ROC) curve values (up to 0.9979) for both sets of data are comparable. The present study shows significant differences between normal samples and each of malignant and benign samples. Only subtle differences exist between malignant and benign lyophilized breast tissue samples where FWHM=0.7±0.1 and 0.8±0.3, AUC=1.3±0.2 and 1.4±0.2 and I1/I2%=44.9±11.0 and 52.4±7.6 for malignant and benign samples respectively.

Proto-oncogene PIM-1 is a novel estrogen receptor target associating with high grade breast tumors

We searched ERα cistromes of MCF-7 breast cancer cells for previously unrecognized ERα targets and identified proto-oncogene PIM-1 as a novel potential target gene. We show that the expression of PIM-1 is induced in response to estradiol in MCF-7 cells and that the induction is mediated by ERα-regulated enhancers located distally upstream from the gene. In keeping with the growth-promoting role of the PIM-1, depletion of the PIM-1 attenuated the proliferation of the MCF-7 cells, which was paralleled with up-regulation of cyclin-dependent protein kinase inhibitor CDKN1A and CDKN2B expression. Analysis of PIM-1 expression between invasive breast tumors and benign breast tissue samples showed that elevated PIM-1 expression is associated with malignancy and a higher tumor grade. In sum, identification of PIM-1 as an ERα target gene adds a novel potential mechanism by which estrogens can contribute to breast cancer cell proliferation and carcinogenesis.

Imaging of human breast tissue using polarization sensitive optical coherence tomography

We report a study on the use of polarization sensitive optical coherence tomography (PSOCT) for discriminating malignant (invasive ductal carcinoma), benign (fibroadenoma) and normal (adipocytes) breast tissue sites. The results show that while conventional OCT, that utilizes only the intensity of light back-scattered from tissue microstructures, is able to discriminate breast tissues as normal (adipocytes) and abnormal (malignant and benign) tissues, PS-OCT helps in discriminating between malignant and benign tissue sites also. The estimated values of birefringence obtained from the PSOCT imaging show that benign breast tissue samples have significantly higher birefringence as compared to the malignant tissue samples.

Transcriptional Analysis of Novel Hormone Receptors PGRMC1 and PGRMC2 as Potential Biomarkers of Breast Adenocarcinoma Staging

The expression of progesterone receptor membrane component 1 (PGRMC1) in breast cancer has generated interest in this recently discovered protein because of its role in tumorigenesis. However, correlations between patient age, PGRMC1 gene expression, breast cancer morphology, and breast cancer stage have not been adequately studied. Furthermore, very little is known about possible roles for other PGRMC isoforms in breast cancer, like PGRMC2. Thus, we examined the expression of PGRMC1 and PGRMC2 mRNA by relative quantitative PCR (RelqPCR) and determined whether transcript levels correlate with age, breast cancer staging, estrogen receptor alpha (ERα) status, and other morphometric features routinely used during the pathological examination of breast ductal adenocarcinomas. Twenty-eight frozen or paraffin embedded breast cancer samples (ductal carcinoma in situ and stages I thru IV invasive ductal adenocarcinoma) and 10 control benign breast tissue samples were randomly selected and interrogated by RelqPCR to determine PGRMC1, 2, and ERα mRNA transcript levels. To control for slight variations in sample preparation, receptor transcript was normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1). Descriptive statistics and ANOVA of multiparametric datasets were used to correlate transcript levels with pathological staging parameters. PGRMC1 mRNA levels decreased significantly with patient age (Pearson's correlation -0.369; P=0.035), whereas PGRMC2 levels did not. Although the mean relative expression of PGRMC1 significantly decreased in stage II breast cancer compared with controls (P=0.050), it was no longer significant when age was considered a covariance (P=0.371). On the other hand, PGRMC2 mRNA transcript was significantly decreased in stage II breast cancer when compared to stage III cancer (P=0.028) in a manner independent of age (corrected model Bonferroni pair wise comparison, P=0.036). Furthermore, PGRMC2 levels positively correlated with ERα mRNA transcripts in patients with ER positive tumors (Pearson's correlation 0.503, P=0.096). Decreases in PGRMC1 mRNA are partially explained by increasing patient age. On the other hand, compared to stage III, PCRMC2 mRNA was significantly decreased in stage II adenocarcinoma of the breast in an age-independent manner. Additionally, PGRMC2 mRNA levels displayed a positive correlation with ERα transcripts. Thus, in addition to morphometric pathologic staging criteria, measurements of PGRMC2 mRNA may be useful for distinguishing low stage tumors from higher stages that require more aggressive clinical management, and may be a useful test when tumor ER IHC results are equivocal.

Mammographic density and epithelial histopathologic markers

BackgroundWe explored the association of mammographic density, a breast cancer risk factor, with hormonal and proliferation markers in benign tissue from tumor blocks of pre-and postmenopausal breast cancer cases.MethodsBreast cancer cases were recruited from a case-control study on breast density. Mammographic density was assessed on digitized prediagnostic mammograms using a computer-assisted method. For 279 participants of the original study, we obtained tumor blocks and prepared tissue microarrays (TMA), but benign tissue cores were only available for 159 women. The TMAs were immunostained for estrogen receptor alpha (ERα) and beta (ERβ), progesterone receptor (PR), HER2/neu, Ki-67, and Proliferating Cell Nuclear Antigen (PCNA). We applied general linear models to compute breast density according to marker expression.ResultsA substantial proportion of the samples were in the low or no staining categories. None of the results was statistically significant, but women with PR and ERβ staining had 3.4% and 2.4% higher percent density. The respective values for Caucasians were 5.7% and 11.6% but less in Japanese women (3.5% and -1.1%). Percent density was 3.4% higher in women with any Ki-67 staining and 2.2% in those with positive PCNA staining.ConclusionThis study detected little evidence for an association between mammographic density and expression of steroid receptors and proliferation markers in breast tissue, but it illustrated the problems of locating tumor blocks and benign breast tissue samples for epidemiologic research. Given the suggestive findings, future studies examining estrogen effects in tissue, cell proliferation, and density in the breast may be informative.

Insulin-Like Growth Factor Binding Protein-2 Is a Novel Therapeutic Target Associated with Breast Cancer

Insulin-like growth factor (IGF) binding proteins (IGFBP) modulate interactions of IGF ligands with the IGF-I receptor. The role of IGFBPs, and specifically IGFBP-2, in breast cancer progression has been poorly defined. This study assesses the effect of IGFBP-2 on the behavior of human breast cancer using clinical specimens as well as in vitro and in vivo experimental systems. 4,181 primary invasive breast cancers and 120 benign breast tissue samples were identified for tumor tissue microarray construction and immunostained with IGFBP-2 antibody. Estrogen receptor-negative MDA-MB-231 cells constitutively overexpressing IGFBP-2 (MDA-MB-231BP-2) were created to assess the effect of IGFBP-2 gain-of-function. MDA-MB-468 cells, naturally expressing IGFBP-2, were used to determine the effect of IGFBP-2 loss-of-function using OGX-225, an antisense oligonucleotide drug candidate. IGFBP-2 expression was significantly higher in breast cancer tissue compared with benign breast tissue. MDA-MB-231BP-2 cells grew more rapidly and were more resistant to paclitaxel both in vitro and in vivo compared with parental cells. OGX-225 decreased IGFBP-2 expression and attenuated the associated aggressive phenotype of MDA-MB-231BP-2 cells both in vitro and in vivo. Furthermore, OGX-225 inhibited the in vitro and in vivo growth of MDA-MB-468 cells. This study provides evidence that IGFBP-2 expression is associated with breast cancer. Novel therapeutics targeting IGFBP-2, such as OGX-225, merit further evaluation.

Promoter hypomethylation of the N-acetyltransferase 1 gene in breast cancer

Arylamine N-acetyltransferase type 1 (NAT1) is reported to be involved in the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs or carcinogens. Gene-specific hypomethylation frequently occurs in a range of cancers and hypomethylation of the genes often correlates well with increased transcription levels. This study was conducted in order to evaluate the methylation status and the transcriptional activity of NAT1 in breast cancer tissues (n=72), benign breast tissues (n=31) and morphologically normal breast tissues (n=30). Our findings showed that the methylation of the NAT1 gene was identified in 39 of the breast carcinomas (54.2%), 23 normal (76.7%) and 25 benign breast tissue samples (80.6%). The breast cancer tissues showed significantly lower methylation rates of the NAT1 promoters than the normal and benign tissues (P=0.012). Furthermore, cancer tissues showed lower methylation density rates than normal and benign breast tissues (P=0.012). The tissues that showed aberrant methylation of NAT1 showed significantly less mRNA expression compared with the unmethylated cases by a thousand fold (P<0.001). Twenty cancers from the methylated group showed positive staining for the estrogen receptor (ER) (51.3%), while 72.7% from the unmethylated group stained positive (P=0.063). Our results suggest that DNA hypomethylation in the NAT1 gene appears to be present in cancerous breast tissues thus indicating that this type of methylation may significantly influence the transcriptional activation of the gene. Therefore, hypomethylation of the NAT1 gene plays a significant role in breast carcinogenesis.

P53 protein accumulation and mutations in normal and benign breast tissue.

Mutations in the p53 gene are amongst the most common molecular changes detected in breast cancer, and there are several reports suggesting that changes in p53 may contribute to the pathogenesis of this disease. In a previous case-control study, we demonstrated that p53 protein accumulation detected by immunohistochemistry in normal or benign breast tissue was associated with a 2.5-fold increase in the risk of subsequent breast cancer. In this study, we investigated whether p53 gene mutations were present in the 29 p53 immunopositive normal or benign breast tissue samples and in 15 p53 immunonegative normal or benign breast tissue samples selected randomly from the original study. DNA was extracted from paraffin sections and underwent PCR-SSCP analysis for exons 4 to 10. PCR products that showed abnormal mobility were excised and sequenced. Sixteen (59.2%) of the 27 immunopositive breast tissue samples and 4 (26.7%) of the 15 immunonegative samples had p53 sequence changes. There was no obvious association between the occurrence of these alterations and any specific histopathologic features. Ten cases showed p53 mutations, and they were all missense base substitutions of the transition type. Thirteen other gene changes occurred in 11 breast tissue samples and consisted of 8 silent (no amino acid change), 4 intronic alterations, and 1 indeterminate alteration. One individual had both a mutation and a silent change. In summary, p53 gene alterations can occur in normal or benign breast tissue, but resolution of their role in the pathogenesis of breast cancer will require long-term follow-up studies involving comparisons of breast cancer occurrence in patients with and without p53 mutations as well as functional assays to determine their significance.

Accumulation of p53 Tumor Suppressor Gene Protein: An Independent Marker of Prognosis in Breast Cancers

Mutations of the tumor suppressor gene p53 have been identified in breast cancer cell lines, and some breast carcinomas are detectable by immunohistochemical assay because of p53 protein accumulation. This study was designed to determine whether p53 protein accumulation in breast cancers correlates with p53 gene mutation, with survival, and with five pathobiologic factors associated with prognosis. IgG1 monoclonal antibody to human p53 protein (PAb 1801) and immunohistochemical methods were used to detect p53 protein accumulation in archival formalin-fixed, paraffin-embedded, randomly selected carcinomas. We studied 295 invasive ductal carcinomas from the Massachusetts General Hospital; 151 were determined to be sporadic (not hereditary). We also studied 97 invasive ductal carcinomas--21 sporadic and 76 familial (hereditary)--from Creighton University. In addition, we examined 31 archival in situ carcinomas, 15 snap-frozen invasive ductal carcinomas, primary cell cultures from three benign breast tissue samples, and breast carcinoma cell lines MDA-MB-231 and MDA-MB-468. Nuclear p53 protein was observed in 16% of the 31 in situ carcinomas, 22% of the 172 sporadic carcinomas, 34% of the 50 tumors from patients with familial breast cancer, 52% of the 23 tumors from patients with the familial breast and ovarian cancer syndrome, and all three tumors from two patients with the Li-Fraumeni syndrome. There was complete concordance between p53 gene mutation and p53 protein accumulation in the 15 snap-frozen carcinomas and in both breast carcinoma cell lines. Statistically significant associations of p53 protein accumulation with estrogen receptor negativity and with high nuclear grade were found. There were statistically significant associations, independent of other prognostic factors, between p53 protein accumulation and metastasis-free and overall survival, for randomly accrued and for both sporadic and familial tumors. Immunohistochemically detected p53 protein accumulation was an independent marker of shortened survival and was seen more often in familial than in sporadic carcinomas. Our findings also suggest a correlation between p53 protein accumulation and p53 gene mutation.