Malignant phyllodes tumours of the breast display increased stromal p53 protein expression

AIMS: To determine the variation in p53 protein expression in phyllodes tumours and fibroadenomas of the breast. METHODS AND RESULTS: Fifteen phyllodes tumours (six malignant, nine benign) and 20 fibroadenomas were examined for p53 expression by immunohistochemistry. Five of the six malignant phyllodes tumours showed moderate or strong p53 positivity at sites of peri-epithelial stromal condensation and atypia. All 20 fibroadenomas, nine benign phyllodes tumours and one malignant phyllodes tumour showed either negativity or focal weak nuclear positivity of scattered stromal cells. CONCLUSIONS: Increased p53 immunoreactivity is present in malignant phyllodes tumours in contrast to benign phyllodes tumours and fibroadenomas. Malignant phyllodes tumours display a distinctive pattern of p53 immunostaining which may be of diagnostic value. These findings suggest that p53 protein may be important in the progression of benign to malignant phyllodes tumours.     

The expression of p53 and bcl-2 in gastrointestinal stromal tumours is associated with anatomical site, and p53 expression is associated with grade and clinical outcome

AIMS: To compare the expression of p53 and bcl-2 in gastrointestinal stromal tumours (GISTs) with anatomical site, National Institutes of Health (NIH) risk category (grade), pathological features, and clinical outcome. METHODS AND RESULTS: The immunohistochemical expression of p53 and bcl-2 in 105 GISTs (71 gastric, 20 small intestinal, four colonic, 10 rectal) was recorded. When all GISTs were assessed, there was p53 positivity in 28% and bcl-2 positivity in 77%. Gastric tumours had a lower prevalence of p53 positivity (20%) than intestinal (40-50%). Rectal GISTs had the lowest prevalence of bcl-2 positivity (20%) and gastric and small intestinal the highest (80% and 90%, respectively). In GISTs from all sites, p53 positivity was associated with size > 50 mm, epithelioid cell shape, nuclear atypia, mucosal invasion, and mitotic count > 5/50 high-power fields. In gastric GISTs the associations were the same, apart from size and mitotic count. In GISTs from all sites and in gastric GISTs, p53 expression correlated with NIH risk category. When GISTs from all sites were subjected to univariate survival analysis, an adverse outcome was associated with p53 positivity, NIH risk category, and several established prognostic factors. When gastric GISTs were assessed, the associations were similar although size was not prognostic. In multivariate survival analysis, p53 expression was independently prognostic for gastric GISTs in some models, while it was never independently prognostic for GISTs from all sites. Whether all GISTs or gastric GISTs were assessed, bcl-2 showed no association with clinical outcome or risk category. CONCLUSIONS: Anatomical site influences p53 and bcl-2 expression in GISTs. p53 expression is associated with NIH risk category, various pathological features, and clinical outcome, and may be independently prognostic for gastric GISTs. Bcl-2 expression has no prognostic value.     

bcl-2 is preferentially expressed in tumours of muscle origin but is not related to p53 expression

bcl-2 is a proto-oncogene which inhibits apoptosis. In contrast, p53 tumour suppressor gene is known to induce apoptosis. In this study we analysed immunohistochemically 63 mesenchymal tumours for the expression of bcl-2 and p53 proteins with a special emphasis on muscle-derived tumours. bcl-2 was expressed in all seven rhabdomyosarcomas and in five out of seven leiomyosarcomas. In benign muscle tumours, bcl-2 expression was found in all four epithelioid leiomyomas and in six out of 14 leiomyomas. In non-neoplastic muscle cells, occasional weak bcl-2 positivity was found in muscle cells of vascular walls and myometrium. In mesenchymal tumours of other lineages, bcl-2 positivity was only found in four out of 12 malignant fibrous histiocytomas and in one out of three liposarcomas. p53 positivity was found in 14 tumours, 13 of which were sarcomas. No association was found between p53 and bcl-2 positivity. Our results suggest that bcl-2 expression is activated significantly more often in muscle derived tumours than in mesenchymal tumours of other lineages. Our results also suggest that p53 status does not directly affect the expression of bcl-2 in sarcomas.     

Immunohistochemical expression of p53 proteins in Wilms' tumour: a possible association with the histological prognostic parameter of anaplasia

Wilms' tumour (nephroblastoma) has been associated with chromosomal abnormalities at the 11p13, 11p15 and 16q regions. A study into the possibility of mutations occurring within p53, the ubiquitous adult tumour suppressor gene, in Wilms' tumour was carried out. Thirty-eight ca ses were studied. Of these 36 were categorised into the favourable histology group and two into the unfavourable histology group based on the National Wilms' Tumour Study criteria. Archival formalin-fixed, paraffin-embedded tissue sections from each case were stained with a polyclonal (AB565:Chemicon) and a monoclonal (DO7:Dako) antibody raised against p53 protein using a peroxidase-labelled streptavidin biotin kit (Dako). 'Cure' (disease-free survival of 60 months or longer) was documente d in 39% of cases with favourable histology tumours. Eleven percent in this group succumbed to the disease. Both cases with unfavourable histology died. Four out of 36 (11%) tumours with favourable histology demonstrated weak to moderate staining with both AB565 and DO7 in more than 75% of tumour cells. In contrast, p53 protein expression in unfavourable histology tumours was significantly increased compared with the favourable histology group ( P  = 0.021) with both cases demons trating immunopositivity in >75% of tumour cells when stained with AB565 and DO7. The intensity of staining ranged from moderate to strong in both cases. It appears from this preliminary study that the immunohistochemical expression of p53 protein in Wilms' tumour, presumably a result of mutation in the p53 tumour suppressor gene, correlates with histological classification, histological categorisation being one of the useful features in the prognostic assessment of Wilms' tumours.     

p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours

Karim R Z, Gerega S K, Yang Y H, Spillane A, Carmalt H, Scolyer R A & Lee C S
(2010) Histopathology 56, 868–875
p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours Aims: Control of cell cycling and proliferation is critical to the development of neoplasia and may play a role in the pathogenesis of phyllodes tumours (PTs). This study aimed to evaluate the immunohistochemical expression of certain proteins from the G₁/S transition of the cell cycle in a cohort of PTs, to determine their role in tumour pathogenesis and to identify any associations with patient outcome. Methods and results: Sixty‐five PTs (34 benign, 23 borderline and eight malignant) diagnosed at a single institution between 1990 and 2006 were analysed. Immunohistochemistry for p16, pRb, cyclin D1 and Ki67 was performed. Expression of the following markers increased significantly with tumour grade: stromal nuclear and cytoplasmic p16 (P = 0.01 and 0.002, respectively), stromal and epithelial pRb (P = 0.000 000 06 and 0.004, respectively), and stromal and epithelial Ki67 (P = 0.03 and 0.04, respectively). Epithelial pRb scores of 7 (range 0–7) were significantly associated with reduced disease‐free survival (DFS) compared with scores of <7 (P = 0.0009). No relationship was found between cyclin D1 expression in either the epithelium or the stroma, and grade or DFS. Conclusions: The results suggest that alterations at the G₁/S transition of the cell cycle play an important role in the progression of PTs.     

p53 expression is common in malignant mesothelioma

The p53 tumour suppressor gene has been shown to be frequently mutated in a wide range of human neoplasms. This is accompanied by increased levels of p53 protein which become immunologically detectable in pathological material. We have investigated the possibility that the differential diagnosis between reactive and neoplastic mesothelium might be resolved using a polyclonal serum raised to human p53 protein, CM-1. None of 20 cases of reactive mesothelial proliferation showed p53 immunoreactivity while 70% (14 of 20) of cases of malignant mesothelioma showed p53 staining. We can thus infer that abnormalities of p53 appear to be a common event in malignant mesothelioma and that p53 immunostaining may be of value in the distinction of malignant mesothelioma from reactive hyperplasia.     

Aberrant expression of the tumour suppressor gene p53 is very frequent in Wilms' tumours.

A series of 34 Wilms' tumours have been analysed for abnormal expression of the tumour suppressor gene p53 using frozen section immunohistochemistry. All tumours showed immunoreactivity with at least one of the specific antibodies used (monoclonal antibody PAb240, polyclonal antibodies CM1 and JG8). Abnormalities of p53 expression are very frequent in this type of childhood tumour.     

Absence of overexpression of p53 protein by intestinal carcinoid tumours

A recessive gene on chromosome 17 encodes a protein, known as p53, which normally acts to regulate the cell cycle, its mutation and overexpression being amongst the commonest genetic abnormalities in human malignant neoplasms. As detected by immunolabelling using the anti-p53 protein antibody D07, overexpression was absent from a series of 22 intestinal carcinoid tumours (ten ileal, nine appendiceal, and three colorectal), nine overtly malignant, but was readily demonstrable in five of five colorectal adenocarcinomas, five of six cloacogenic carcinomas, and four of five squamous carcinomas of the anal canal used as controls. These observations are in keeping with previous similar studies of pulmonary carcinoid tumours and suggest possible differences in the pathogenesis of such neoplasms in comparison with non-endocrine differentiated tumours arising at equivalent sites.     

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx

p21^WAF1/Cip1 is a recently identified gene involved in cell cycle regulation through cyclin-CDK-complex inhibition. The expression of this gene in several cell lines seems to be induced by wild-type, but not mutant, p53. p21^WAF1/Cip1 expression has been studied at both mRNA and protein levels in a series of 49 normal mucosae and squamous cell carcinomas of the larynx. A significant association was found between mRNA and protein expression in tumours (P<0·0001). p21^WAF1/Cip1 expression was strongly associated with squamous cell differentiation of carcinomas, because six of seven (86 per cent) undifferentiated carcinomas (grade 4) showed very low levels of p21^WAF1/Cip1 expression, whereas 41 out of 42 (98 per cent) carcinomas with squamous cell differentiation (grades 1–3) had normal or high levels of p21^WAF1/Cip1 expression (P<0·0001). In addition, p21^WAF1/Cip1 expression was topologically related to the squamous differentiation of tumour cells with a distribution similar to that seen in normal squamous epithelium. No correlation was found between p21^WAF1/Cip1 expression and the global S-phase of the carcinomas. p53 mutations (exons 5–9) were found in ten carcinomas with p21^WAF1/Cip1 expression, but no p53 mutations were detected in three p21^WAF1/Cip1-negative tumours. In conclusion, p21^WAF1/Cip1 expression is frequently upregulated in squamous cell carcinomas of the larynx and is associated with tumour cell differentiation. p21^WAF1/Cip1 expression in these tumours is independent of p53 gene mutations. © 1997 John Wiley & Sons, Ltd.     

p53 but not erbB-2 expression is associated with rapid tumor proliferation in urinary bladder cancer

Tumor proliferation in bladder cancer is associated with tumor behavior. To assess the association between Ki-67 labeling index (LI), p53, and c-erbB-2 overexpression, formalin-fixed tissue samples of 160 patients with transitional cell carcinoma (TCC) of the urinary bladder were studied by immunohistochemistry. Ki-67 LI was strongly associated with tumor stage (P < .0001), tumor grade (P < .0001), and p53 status (P = .0014) but not with erbB-2 overexpression (P > .2). Ki-67 LI was higher in p53-positive tumors (19%) than in p53-negative tumors (14%) when all stages were compared. Ki-67 LI was independent of p53 expression in pTa tumors (p53-positive, 9%; p53-negative, 11%), showing that p53 overexpression alone is not sufficient to induce rapid tumor cell proliferation in pTa tumors. Ki-67 LI also was independent of p53 expression in pT2 to pT4 tumors (p53-positive, 20%; p53-negative, 23%), indicating that p53 expression is not necessary for rapid tumor cell proliferation in advanced stages. However, there was a striking difference in Ki-67 LI between p53-positive pT1 tumors (22.0% ± 8.8 standard deviation [SD]; n = 20) and p53-negative pT1 tumors (9.7 ± 8.3 SD; n = 22; P = .0001). These results suggest that increased proliferation in p53-positive pT1 tumors is caused by additional alterations that occur during tumor progression.     

p53 but not bcl-2 is expressed by most cholangiocarcinomas: a study of 28 cases

AIMS: To examine the frequency and pattern of expression of p53 and bcl-2 in archival material from patients with cholangiocarcinomas and to evaluate their respective roles in its pathogenesis, diagnosis and prognosis. METHODS AND RESULTS: Twenty-eight surgical cases of cholangiocarcinomas diagnosed at St James's University Hospital and 16 control cases were immunostained with monoclonal antibodies to p53 and bcl-2 using streptavidin-biotin complex method. Pressure cooker was used for antigen retrieval. Of the cholangiocarcinomas, 85.7% (24/28) overexpressed p53. The intensity of staining in these cases varied from 1+ in 2, 2+ in 10 and 3+ in 12 cases. None of the 28 tumours expressed bcl-2. The well differentiated nature of the tumour made assessment of dysplasia difficult, however, where present it did not express p53 or bcl-2. The bile duct epithelium adjacent to the tumour and in the control cases did not show any significant nuclear staining for either antigen. CONCLUSIONS: Overexpression of p53 appears to play an important role as a late event in the pathogenesis of cholangiocarcinomas, while we found no evidence of bcl-2 overexpression. The expression of p53 in 86% of the invasive tumours, as compared to its lack in the adjacent normal bile duct epithelium, makes it potentially useful in the diagnostic histopathology of these cases.     

P53 mutation does not affect prognosis in ovarian epithelial malignancies

Mutations of the p53 tumour suppressor gene have been found in most human cancers, including ovarian epithelial malignancies. This study investigated whether the presence or absence of p53 mutation was associated with outcome following platinum-based chemotherapy in patients with ovarian cancer. DNA samples from tumour tissue and blood were obtained from 73 patients with primary tumours, 50 of whom received platinum-based adjuvant chemotherapy. Single-strand conformation polymorphism analysis and direct DNA sequencing of exons 5-8 detected mutations in 44% (32 of 73) of tumours. These were more common in late-stage (III or IV) than in early-stage disease (I or II) (p=0.03). There was no association with histological type, volume of residual disease following surgery, or initial CA125 levels. No significant association was found between p53 status and overall survival or disease-free survival following chemotherapy. Likewise, there was no correlation between p53 mutation and response to chemotherapy as defined by normalization of CA125 levels. Tumours with p53 missense mutations recurred within a significantly shorter time than those with normal p53 (p=0.04). In addition, there was a tendency for tumours with missense mutations to have a shorter disease-free survival than those with non-missense mutations, although this did not reach statistical significance (p=0.07).     

Overexpression of the tumour suppressor gene p53 is not implicated in neuroendocrine tumour carcinogenesis

The tumorigenesis of neuroendocrine tumours remains poorly understood, although a minority, the familial multiple endocrine neoplasia (MEN 1 and MEN 2), are known to be of uncommon genetic origin. Mutation of the tumour suppressor gene, p53, is now known to be a common genetic alteration in about half of all types of non-endocrine cancers. In the present study, immunocytochemistry using the monoclonal anti-p53 antibody, DO-7, has been employed to investigate the accumulation of p53 immunoreactivity in a wide range of primary neuroendocrine tumours. Tumours (n=109) were fixed and processed to paraffin wax according to a constant protocol. Sections were subjected to microwave antigen retrieval prior to immunostaining for p53. Positive nuclear immunostaining was obsered in one medullary carcinoma of the thyroid (MCT), one lung carcinoid, and five small cell carcinomas of the lung (SCCL). All other tumour samples were consistently negative. As the neoplasia investigated in this study comprised a wide spectrum of neuroendocrine tumour types and ranged from minute, relatively benign lesions to malignant metastasizing disease and as there was no relationship between the presence of p53 overexpression and clinico-pathological features, the present study suggests that p53 gene mutation may be relatively unimportant in the genesis of neuroendocrine tumours.     

p63 expression in ovarian tumours: a marker for Brenner tumours but not transitional cell carcinomas

AIMS: To investigate p63 expression in ovarian neoplasms. METHODS AND RESULTS: Immunohistochemistry using an antibody that detects all p63 isoforms was performed on 103 primary ovarian neoplasms of different histological types. Diffuse nuclear immunoreactivity of p63 was demonstrated in the 17 benign and five borderline Brenner tumours. Only one of the six malignant Brenner tumours displayed p63 expression. p63 immunoreactivity was absent in all the ovarian transitional cell carcinomas (TCC), but was demonstrated extensively in TCCs of the urinary bladder. Besides focal p63 expression in epidermal basal cells of immature and mature teratomas, all other ovarian lesions were devoid of p63 expression. p63 expression was also demonstrated in cervical transitional cell metaplasia and Walthard cell nests of fallopian tubes. CONCLUSIONS: Expression of p63 protein is apparently cell lineage specific and in ovarian neoplasms is confined to benign and borderline Brenner tumours. The loss of expression in malignant Benner tumours suggests a role for p63 in Brenner carcinogenesis. The distinct patterns of p63 expression in TCCs in the ovary and urinary bladder may help in their differential diagnosis.     

A unique basal pattern of p53 expression in ulcerative colitis is associated with mutation in the p53 gene

AIMS: The p53 protein is implicated in the control of cell proliferation, differentiation, and death. As part of a study characterizing p53 alterations in colonic mucosa of patients with ulcerative colitis, we identified a unique pattern of basal p53 immunoreactivity. METHODS AND RESULTS: Tissue samples (n=180) from 42 ulcerative colitis patients were evaluated for p53 alterations by immunohistochemistry, loss of heterozygosity analysis, polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. In addition, the expression of the p53- associated proteins p21waf1/cip1 and MDM2 was evaluated immunohistochemically. Three basic patterns of p53 immunoreactivity were observed: (i) isolated immunoreactive cells in the crypt bases; (ii) strongly positive cells confined to the basal half of the glands; and (iii) diffusely staining cells. The basal staining pattern was observed in both non-neoplastic tissues and in some areas of dysplasia, and was associated with normal expression of p21waf1/cip1 in all cases, and with p53 mutation in seven of 11 cases. CONCLUSIONS: The basal pattern of p53 expression is associated with mutation in the p53 gene, and appears to be an early change in a subgroup of ulcerative colitis patients. The significance of this pattern of immunoreactivity and the mechanism by which it develops are discussed.     

Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.     

Loss of p53 partially rescues embryonic development of Palb2 knockout mice but does not foster haploinsufficiency of Palb2 in tumour suppression

PALB2 interacts with BRCA1 and BRCA2 in supercomplexes involved in DNA repair via homologous recombination. Heterozygous germline mutations in PALB2 confer a moderate risk of breast cancer, while biallelic PALB2 mutations are linked to a severe form of Fanconi anaemia characterized by early childhood solid tumours and severe chromosomal instability. In contrast to BRCA1- or BRCA2-associated cancers, breast tumours in heterozygous PALB2 mutation carriers do not show loss of the wild-type allele, suggesting PALB2 might be haploinsufficient for tumour suppression. To study the role of PALB2 in development and tumourigenesis, we have generated Palb2(GT) mouse mutants using a gene trap approach. Whereas Palb2(GT/GT) homozygous mutant embryos died at mid-gestation due to massive apoptosis, Palb2(GT/+) heterozygous mice were viable and did not show any obvious abnormalities. Deletion of p53 alleviated the phenotype of Palb2(GT/GT) embryos, but did not rescue embryonic lethality. In addition, loss of p53 did not significantly collaborate with Palb2 heterozygosity in tumourigenesis in heterozygous or homozygous p53 knockout mice. Tumours arising in Palb2(GT/+) ;p53(+/-) or Palb2(GT/+) ;p53(-/-) compound mutant mice retained the wild-type Palb2 allele and did not display increased genomic instability.