Nuclear Accumulation of p53 Protein Correlates with Mutations in the p53 Gene on Archival Paraffin-embedded Tissues of Human Breast Cancer

Fifty invasive ductal carcinomas of the breast were analyzed by immunohistochemistry, polymerase chain reaction-single strand conformationl polymorphism (PCR-SSCP) and direct sequencing after microdissection of conventional formalin-fixed, paraffin-embedded tissues. A highly significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found ( P < 0,0001). Of 13 tumors that demonstrated p53 gene mutations, 11 (84.6%) showed nuclear accumulation of p53 protein. However, of 37 tumors in which gene mutations were not detected, only 5 (13.5%) showed nuclear accumulation of p53 protein. There was a statistically significant association between the nuclear accumulation of p53 protein and a higher histological grade ( P < 0.001) or mitotic index ( P <0.01). In addition, gene mutations had a statistically significant association with a higher histological grade ( P <0.05) or mitotic index ( P > 0.0001). Therefore, p53 abnormalities might be associated with an aggressive phenotype in breast cancer. We conclude that the immunohistochem-ical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in archival paraffin-embedded tissues, and that this method is useful for rapid screening of p53 abnormalities.     

Japanese late-onset breast cancer families: Their clinicopathological characteristics and absence ofBRCA1 andBRCA2 germline mutations

BACKGROUND: The causes and pathologic and prognostic phenotypes of late-onset familial breast cancers are still unknown. The purpose of this study was to document the clinicopathological features of late-onset familial breast cancers using genetic testing ofBRCA1 andBRCA2. METHODS: We analyzed 11 breast cancers from 10 patients from 8 Japanese late-onset Breast cancer families. RESULTS: The average age of the patients was55 years (range 43 to 89). Bilateral occurrence was noted in 2 patients (8%). All the tumors were invasive ductal carcinomas, except for 1 case of invasive lobular carcinoma. Tumor size ranged from 0.8 cm to 7.8 cm (median 2.3 cm) and lymph node metastasis occurred in 6 of the 11 patients (55%). Six (55%) of the 11 tumors were histologically grade 2 and 5 (45%) were histologically grade 3. Estrogen receptor (ER) positivity was 80% (8/10). Overexpression of c-erbB-2 and p53 protein was detected in 18% (2/11) and 9% (1/11) of the tumors, respectively. Five patients from 4 families received genetic testing but all were negative forBRCA1 andBRCA2 germline mutations. All the patients were alive after a median follow-up period of 32 months, except for 1 patient. CONCLUSIONS: In this study, no germline mutations ofBRCA1 orBRCA2 were detected. However, there was a tendency towards ER-positive tumors, but the positivity of p53 protein was considered to be lower then that of sporadic tumors.     

P53 and C-erbb-2 alterations in in-situ and invasive ductal breast carcinomas - a genetic and immunohistochemical analysis

p53 mutations, c-erbB-2 amplifications and expression of the related proteins were evaluated in a panel of ductal breast carcinomas selected on the basis of their invasive component. The tumors comprised: 8 ductal carcinomas in situ (DCIS); 8 carcinomas with a minimal (less than 20%) invasive component, hereafter referred to as DCIC (<20%); 13 carcinomas with 20%-50% invasiveness, DCIC (20-50%), and 48 infiltrating carcinomas with more than 50% invasive component, DCIC (>50%). Tumors were further subdivided into large pleomorphic cell type or small regular cell type. A strong association was present between p53 gene mutations and p53 protein overexpression (p<0.001) as well as between amplification of the c-erbB-2 gene and expression of its protein product (p=0.006). p53 aberrations (gene mutation and/or protein overexpression) were observed in 1 (12%) of 8 DCIS, 1 (11%) of 9 DCIC (<20%), 3 (23%) of 13 DCIC (20%-50%), and 13 (28%) of 47 DCIC (>50%). Amplification and/or overexpression of c-erbB-2 were found in 30 (39%) of the 77 breast carcinomas analyzed and were more frequent in DCIC (<20%) and in DCIC (20%-50%) (56% and 46% respectively) than in DCIS or DCIC (>50%) (12% and 38% respectively). Irrespective of the presence of invasion, tumors with p53 or c-erbB-2 alterations showed more frequently large cells with pleomorphic nuclei, (for p53, p=0.027; for c-erbB-2, p=0.014). Our data suggest that p53 and c-erbB-2 alerations may occur in the earliest recognized phase of breast cancer and may be important in the evolution of small cell to large cell mammary carcinoma during tumor progression.     

Aberrant cytoplasmic expression of the p16 protein in breast cancer is associated with accelerated tumour proliferation.

The p16 protein plays an important role in the transition of cells into the G1 phase of the cell cycle. We have studied the prevalence of p16 protein expression in breast carcinomas in a prospective series of 368 invasive and 52 non-invasive malignancies, as well as in 88 locally recurring tumours and three tumour cell lines. p16 protein expression was evaluated immunohistochemically on paraffin sections using monoclonal and polyclonal anti-p16 antibodies, and by immunoblotting of tumour cell suspensions. Tumour cell lines were also subjected to polymerase chain reaction-single strand polymorphism (PCR-SSCP) analysis and direct DNA sequencing. The results were compared with established prognostic parameters, DNA flow cytometry and p53 protein expression. In 33 (9%) invasive and two (4%) intraductal carcinomas, a cytoplasmic accumulation of the p16 protein was seen. These cases were characterized by poor histological grade of differentiation, loss of of oestrogen receptors and progesterone receptors and frequent overexpression of the p53 protein. In addition, breast carcinomas with aberrant p16 expression demonstrated a high proliferative activity, with median S-phase fractions 74% higher than in the control group and the median Ki67 fractions elevated to 75%. A genetic alteration of the p16 gene was not detectable in three analysed cell lines with cytoplasmic p16 expression applying PCR-SSCP and direct DNA sequencing. These results indicate that cytoplasmic accumulation of the p16 protein identifies a subset of highly malignant breast carcinomas with accelerated tumour proliferation and other unfavourable parameters in breast cancer. The described protein accumulation is apparently not caused by an alteration of the p16 gene.     

Expression of bax and p53 proteins in the tumorigenesis and progression of breast carcinomas.

OBJECTIVES: Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis of breast cancer. The purpose of this study was to investigate the role of bax and p53 expression in tumorigenesis and progression of breast carcinoma as well as their relationship with proliferative and apoptotic activity. METHODS: We used immunohistochemical methods and in situ detection of apoptotic cells to examine 30 carcinomas in situ (CIS), 131 invasive breast carcinomas and 45 lymph node metastases. RESULTS: In 25% (33 of 131) of invasive breast carcinomas examined, bax expression was absent, while p53 accumulation was present in 37% (49 of 131). Interestingly, p53 accumulation and loss of bax expression occur in breast CIS as frequently as in invasive breast carcinoma. Thus, in 17% (5 of 30) of CIS bax expression was absent, and 30% (9 of 30) presented nuclear expression of p53. p53 accumulation was related to apoptosis and proliferative activity. However, the protein level of bax was unrelated to all parameters studied, including proliferation and apoptosis of tumor cells. A multivariate analysis of disease-free survival demonstrated that p53 accumulation and bax expression are not significant independent indicators of prognosis in operable breast carcinoma. Our results also show that the proportion of bax- and p53-positive cells does not vary between primary and metastatic tumors. CONCLUSIONS: p53 accumulation and loss of bax expression influence the acquisition of a malignant phenotype but seem to have no further impact on tumor progression.     

p53 Protein Accumulation in Non-Invasive Lesions Surrounding p53 Mutation Positive Invasive Breast Cancers

p53 mutation is a common event in sporadic breast cancer being found in 15–50% of invasive carcinomas. The purpose of this study was to determine the earliest histologic stage at which p53 mutation could be detected with a widely used anti-p53 antibody (DO7, Novocastra) which recognizes both wild type and mutant forms. p53 expression was assessed immunohistochemically in 12 primary breast carcinomas with known p53 mutations and in all pre-malignant epithelial lesions surrounding these invasive cancers. Strong p53 nuclear staining was found in all of the tumors known to have missense mutations and none of the tumors with truncation mutations. In cases with intense staining in the invasive carcinoma, a similar quality of staining was also seen in all areas of DCIS (ductal carcinoma in situ) and was representative of missense p53 mutations. Lighter nuclear staining intensity was observed in up to 40% of cells in areas of hyperplasia and in up to 30% of normal breast lobules irrespective of the type of mutation found in the invasive carcinoma. This weak staining was not specific to mutated p53 and may indicate increased amounts of normal p53 protein.We conclude that p53 inactivation occurs prior to invasion in breast carcinogenesis, with mutations being uniformly identified in DCIS associated with p53-mutated invasive carcinomas. In contrast, there is no evidence that epithelial hyperplasia or epithelial cells of the terminal duct lobular unit harbor the same mutations as their associated invasive carcinoma.     

人乳腺癌细胞株WAF1／GIP1／p21基因的表达及其与细胞生物学特性的关系

OBJECTIVE: To study the WAF1/CIP1 gene DNA status, mRNA and protein expressions in human breast cancer cell line and its significance. METHODS: Using cell culture, molecular biological techniques such as Southern blot, Northern blot and immunocytochemical methods, the WAF1/CIP1 gene DNA status, mRNA and protein expression levels in MCF-7 expressing wild type p53(wtp53) and MDA-MB-231 expressing mutant p53 (mtp53) human breast cancer cell lines were detected respectively. The p53 and mdm-2 protein expression levels and cytobiological features of the 2 cell lines were compared and correlated to their WAF1/CIP1 gene expression levels. RESULTS: (1) There was no difference in WAF1/CIP1 gene DNA status in the two breast cancer cell lines. Neither of them showed gene amplificatian or deletion. However, the WAF1/CIP1 mRNA and p21WAF1/CIP1 protein expression levels of MCF-7 cells were higer than those of MDA-MB-231 cells (P < 0.05). (2) The character and cellular distribution of p53 protein in the two cell lines were clearly different. The expression level of mdm-2 proteion was significantly higher in MCE-7 than in MDA-MB-231 cells (P < 0.05). (3) Compered to the other breast cancer cell line, MCF-7 cells were better differentiated, grew more slowly and adhered more closely with each other. CONCLUSION: The WAF1/CIP1 gene expression at mRNA and protein levels is associated with p53 phenotype and some cytobiological features of human breast cancer.     

p53 Inactivation is a Rare Event in Familial Breast Tumors Negative for BRCA1 and BRCA2 Mutations

Germline mutations at BRCA1 or BRCA2 genes result in susceptibility to breast and ovarian cancers. BRCA1- and BRCA2-associated tumors have distinct histologic and molecular phenotypes, as compared to sporadic breast tumors. Typically, a higher grade of malignancy is observed in BRCA-associated cancers. A number of studies have suggested that BRCA1 and BRCA2 proteins are of importance in DNA repair and maintenance of genome integrity, bringing about molecular models of tumor pathogenesis. In particular, alterations at p53 gene have been suggested to be a necessary step in the tumorigenesis of BRCA-associated carcinomas. In fact, BRCA-associated breast cancers have higher p53 mutation frequencies than sporadic ones. At present, very little is known regarding BRCA non-associated familial tumors (termed BRCAx tumors). To our knowledge no data is available on p53 alterations in this sub-group of familial tumors. In this study p53 alteration frequencies were evaluated in 13 BRCA1, 11 BRCA2 and 55 BRCAx breast tumors. Tumor samples were analyzed for p53 gene mutations by PCR-SSCP/direct sequencing, and for p53 protein overexpression by immunohistochemistry (IHC). Altogether, p53 alterations were detected in 54% of BRCAI tumors compared with 5% of BRCAx tumors. No p53 alteration was found in BRCA2 tumors. While loss of p53 checkpoint control is likely to be an important step in the molecular pathogenesis of BRCA1-associated cancers, our data seem to indicate a p53-independent molecular mechanism underlying BRCAx neoplastic transformation.     

p63 expression in normal,hyperplastic and malignant breast tissues

BACKGROUND: p63 is a homologue of the p53 tumor suppressor gene and its protein is selectively expressed in the basal cells of a variety of epithelial tissues. It has recently been confirmed that p63 is expressed in the basal cells of normal prostate glands but not in prostatic carcinomas. Whether expression of p63 in breast correlates with tumor progression is the focus of this study. METHODS: Forty cases, which all contained normal breast tissue, ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma in the same patient were included in this investigation using an indirect immunohistochemical method and double staining. RESULTS: p63 was exclusively expressed in the myoepithelial cells of normal breast, partially expressed in ductal hyperplasia, rarely expressed in carcinoma in situ and not expressed in invasive carcinomas. CONCLUSION: The results suggest an association between loss of p63 expression and progression of breast ductal carcinoma. p63 immunostaining might be of assistance for distinguishing invasive ductal carcinoma from ductal carcinoma in situ or rare questionable ductal hyperplastic lesions, leading to correct therapy clinically.     

Expression of c-erbB3 protein in primary breast carcinomas.

Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer.     

Histological classification of invasive ductal carcinoma and the biological parameters in breast cancer

BACKGROUND: The histological classification of invasive ductal carcinoma proposed by the Japanese Breast Cancer Society is based on the appearance of breast cancer invasion. However, few studies have been done to clarify the relationship between the histological classification and other parameters that represent the biological characteristics of individual breast cancer cells. MATERIALS AND METHODS: We analyzed 1,025 invasive ductal carcinomas, consisting of 424 papillotubular, 330 solid-tubular and 271 scirrhous cases, with regard to the status of estrogen receptor (ER), progesterone receptor (PgR), DNA ploidy, epidermal growth factor receptor (EGFR) and p53 protein. RESULTS: Absence of ER and PgR, aneuploidy, EGFR positivity and p53 protein positivity were all observed significantly more frequently in solid-tubular tumors than in the other two types. In addition, the number of abnormal features with regard to these 5 biological parameters was also significantly higher in solid-tubular tumors than in the other two types. CONCLUSION: Abnormalities in the biological parameters listed above were frequently found in the solid-tubular type of invasive ductal carcinoma.     

p53 protein expression in pancreatic tumors and its relationship to clinicopathological factors and prognosis

We examined the expression of p53 protein by immunohistochemical method in a series of pancreatic tumors and evaluated its relationships to the clinicopathological factors and prognosis. The study involved 108 cases of pancreatic tumors (79 ductal carcinomas, 1 acinar cell carcinoma, 14 endocrine tumors, 6 solid cystic tumors, 8 benign ductal tumors) and 8 chronic pancreatitides. Thirty-nine cases of pancreatic ductal carcinoma (49.4%) were positive for p53 protein. Analysis of the Cox hazards model identified p53 positivity and stage at the initial operation as an independent prognostic factor. Patients with p53 positive ductal carcinomas had a greater risk of death compared to p53 negative cases (P < 0.05). There was, however, no statistically significant correlation between p53 protein expression and other clinicopathological factors. Cases of stage III and IVb with positive p53 showed a bleak prognosis compared to p53 negative cases (P < 0.05). Our results suggest that p53 expression is common in invasive pancreatic ductal carcinomas and may have a prognostic value. © 1996 Wiley-Liss, Inc.     

Prognostic significance of TP53 alterations in breast carcinoma.

Constant denaturant gel electrophoresis (CDGE) was used to screen 179 breast carcinomas for mutations in the conserved regions of the TP53 gene (exons 5 through 8). Mutations were found in 35 of 163 primary tumours (21%) and in 5 of 16 metastases (31%) and resided predominantly in exon 7. The majority of the mutations were G:C-->A:T transitions. Immunohistochemistry demonstrated nuclear accumulation of p53 protein in 35 of 162 primary tumours (22%) and in four of 15 metastases (27%). TP53 mutation was strongly associated with nuclear accumulation of p53 protein. In total 42 of 163 primary tumours (26%) and 5 of 16 metastases (31%) were demonstrated to contain TP53 alterations (mutation and/or nuclear protein accumulation). TP53 alteration in primary tumour was significantly associated with the following parameters: positive node status, T status > 1, negative oestrogen receptor status, negative progesterone receptor status, presence of ERBB2 gene amplification, and invasive ductal histology. Furthermore, there were statistically significant associations, independent of other prognostic factors, between TP53 alterations in primary tumour and disease-free and overall survival.     

Histological grade,p53, HER2 and hormone receptor status of synchronous bilateral breast carcinoma

BACKGROUND AND OBJECTIVES: Histological grade and tumor biology remain important predictors of the clinical behavior of breast carcinomas. We analyzed the clinicopathological characteristics and tumor biology with regard to histological grade (HG), p53, HER2 and hormone receptor status to address this question. PATIENTS AND METHODS: A consecutive series of 74 female synchronous bilateral breast carcinoma patients treated at the National Cancer Center Hospital were the primary source of these retrospective data. Clinicopathological background factors, histological grade and immunohistochemical staining for p53, HER2 and hormone receptor status, were analyzed. RESULTS: Of 148 synchronous bilateral tumors, 102 were invasive ductal carcinoma (IDC). The others included 24 pure or predominant ductal carcinoma in situ (DCIS), 5 spindle cell carcinomas, 16 invasive lobular carcinomas and 1 squamous cell carcinoma. 128 cases (128/148: 89%) were HG 1 (72/148: 49%) or HG 2 (56/148: 38%). The positivity rates for p53, HER2, estrogen receptor (ER) and progesterone receptor (PR) were 9%(14/148), 18%(26/148), 64%(95/148) and 64%(95/148), respectively. CONCLUSION: Our findings indicate that synchronous bilateral breast carcinomas showed a higher frequency of invasive lobular carcinoma, lower HG and higher rate of hormone receptor positivity than unilateral breast carcinomas.     

Two-hit inactivation of FHIT by loss of heterozygosity and hypermethylation in breast cancer.

PURPOSE: The FHIT gene, which spans the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene in breast carcinomas. In this study, we would like to delineate more precisely its role in breast tumorigenesis. EXPERIMENTAL DESIGN: To confirm the tumorigenic role of FHIT, 46 sporadic invasive ductal carcinomas of the breast were tested for the "two hits" required to inactivate this gene. Microsatellite loss of heterozygosity (LOH) was considered as the first hit. To examine the possibility that hypermethylation serves as the second hit for FHIT inactivation, methylation of 5'-CpG islands of FHIT was analyzed by methylation-specific PCR. RESULTS: LOH was detected in 8 of 40 informative tumors, and hypermethylation was observed in 22 of 46 (48%) cases. Aberrant FHIT protein expression was found in 31 of 46 (67%) cases examined. All seven tumors showing both LOH and hypermethylation showed complete loss of Fhit protein expression. In addition, a significant positive association was found between the existence of LOH and 5'-CpG island hypermethylation (P = 0.04), which was consistent with the two-hit model. CONCLUSIONS: To our knowledge, this study provides the first evidence that biallelic inactivation of FHIT by LOH and hypermethylation leads to the complete inactivation of FHIT gene in patients with breast cancer. Silencing of the FHIT gene by promoter hypermethylation occurs in primary breast carcinomas, especially those with LOH. These findings support a role for this tumor suppressor gene in sporadic breast tumorigenesis.     

Elevated frequency and functional activity of a specific germ-line p53 intron mutation in familial breast cancer

Previous studies have determined that the frequency of germ-line p53 mutations in familial breast cancer patients is 1% or less, but these reports have not investigated the importance of polymorphic intron base changes in the p53 gene. Therefore, we investigated the frequency of both exon and intron germ-line p53 base changes in 42 breast cancer patients with a strong family history of breast cancer. The mean age of presentation of these patients was 44.0 years (range, 29-69), and 12 of 42 (29%) were of known Ashkenazi ancestry. Purified DNA obtained from the 42 index cases was screened for germ-line p53 mutations in exons 2-11 and surrounding introns using a combination of intron based primers for PCR-single strand conformation polymorphism analysis, direct sequencing, and microarray sequencing using the Affymetrix p53 gene chip methodology. Morphological analysis of apoptosis and cell survival determination were performed on EBV-immortalized lymphoblastoid cell lines from two patients with the p53 intron 6 mutation. A germ-line mutation in the p53 gene at nucleotide 13964 with a G to C base change (13964GC) was identified in 3 of 42 (7.1%) hereditary breast cancer patients. Two patients were heterozygous for this mutation, and one patient had a homozygous mutation. In comparison, 0 of 171 (0%) of sporadic breast cancer patients had the p53 13964GC mutation (P = 0.0003). We found that 0 of 42 (0%) of these hereditary breast cancer patients had other germ-line p53 mutation in exons 2-11. However, pedigree analysis demonstrated that all three patients had strong family histories of multiple types of cancers consistent with Li-Fraumeni syndrome but with late age of onset. Comprehensive BRCA1 and BRCA2 nucleotide analysis from patients with the p53 13964GC mutation revealed no concomitant deleterious BRCA1 or BRCA2 mutations, although they were found in the other hereditary breast cancer patients. Functional analysis of two immortalized lymphoblastoid cell lines derived from patients with the p53 13964GC mutation demonstrated prolonged in vitro survival in response to cisplatinum treatment and showed decreased chemotherapy-induced apoptosis. Immunohistochemical analysis of breast tumors from these patients revealed high levels of mutant p53 protein, suggesting a functional mutation in the p53 gene. In summary, we have identified a single p53 intron mutation in familial breast cancer patients that is present at elevated frequency and has functional activity.     

TRAF4蛋白在乳腺癌组织中的表达及意义

背景与目的:对肿瘤坏死因子受体相关因子4(tumor necrosis factor receptor-associated factor 4,TRAF4)在乳腺癌中表达的研究存在争议.本研究探讨TRAF4在正常乳腺组织、乳腺癌组织及不同侵袭能力乳腺癌细胞系中的表达情况.方法:应用免疫组化方法检测TRAF4在70例乳腺癌组织和14例正常乳腺组织中的表达.应用Western blot方法检测TRAF4在乳腺癌细胞系MDA-MA-231(高侵袭)和MCF-7(低侵袭)中的表达.结果:TRAF4在正常乳腺组织中呈浆、核阳性表达.其浆阳性率在正常乳腺组织(78.57%)、非浸润性导管癌(88.57%)和浸润性导管癌(91.43%)中逐渐增高,但差异无统计学意义(P＞0.05).而核阳性率在正常乳腺组织(64.28%)中显著高于非浸润性导管癌(28.57%,P＜0.01),在非浸润性导管癌中高于浸润性导管癌(5.7%,P＜0.05).TRAF4总蛋白在乳腺癌高侵袭细胞系中的表达量高于低侵袭细胞系,但差异无统计学意义(P＞0.05).结论:TRAF4在乳腺癌细胞核中表达明显降低,与乳腺癌侵袭性相关.