Multifocal intrafollicular granulosa cell tumor of the ovary associated with an unusual germline p53 mutation.

A 23-year-old woman presented with a 7 cm right multicystic mass in the ovary, which corresponded microscopically to an unusual lesion consisting of a multifocal granulosa cell tumor with intrafollicular ('in situ') growth involving two-thirds of mature follicles. Stromal invasion was found in only one area where neoplastic follicles coalesced. Granulosa cells had atypical, bizarre TP53 positive nuclei with hyperchromatism, abundant mitoses and numerous hyaline globules. The contralateral ovary was normal. From the age of 10 years, the patient had a complex medical history of multiple tumors, including telangiectatic osteosarcoma, typical and malignant phyllodes tumor, reticulohistiocytoma of skin, carcinomas of the breast and lipo- and leiomyosarcoma. The female genital tract also harbored myometrial leiomyomas and an early endometrial carcinoma. Retrospective histologic study of all mesenchymal neoplasms in this patient showed, the conspicuous presence of similar bizarre TP53 positive cells with hyaline globules in all the mesenchymal neoplasms. In the genetic study, a germline p53 gene mutation was detected in exon 10, codon 336, generating a stop codon in the oligomerization domain of the protein (E336X). A further p53 mutation was found in exon 7 in the granulosa cell tumor. Mutation occurred de novo since there was no history of tumors in any family members, all of whom had a wild-type p53. Although this patient shows a typical tumor phenotype of Li Fraumeni syndrome, the germline mutation corresponded to a highly unusual mutated domain, which is similar to the one found in childhood malignant adrenocortical tumor; also a rare neoplasm that originates in adrenocortical cells; which are closely related, both functionally and embryologically, to granulosa cells.     

Retroperitoneal dedifferentiated liposarcoma lacking MDM2 amplification in a patient with a germ line CHEK2 mutation

Germ line mutations in genes that encode proteins involved in the DNA damage response predispose patients to a variety of tumors. Checkpoint kinase 2, encoded by the CHEK2 gene, is important in transducing the DNA damage response. Germ line CHEK2 mutations are seen in a subset of patients with a familial breast cancer and sarcoma phenotype. We report a case of retroperitoneal dedifferentiated liposarcoma in a 61-year-old female with germ line CHEK2 mutation. MDM2 gene amplification normally present and used to aid in the diagnosis of retroperitoneal dedifferentiated liposarcoma was absent in this case. Lack of MDM2 overexpression has similarly been reported in liposarcomas arising in patients with germ line TP53 mutations. We propose this case may highlight a nonamplified MDM2 phenotype in well- and dedifferentiated liposarcomas arising in patients with germ line mutations of genes involved in p53-associated DNA damage response pathways.     

Altered expression of p53 and its regulated proteins in phyllodes tumours of the breast.

The histological characteristics of phyllodes tumours of the breast are often not related to their clinical outcome. Additional studies must therefore be performed to investigate the possible relationship of cell biological parameters to the biological behaviour of these tumours. The expression of Ki-67, p53, and its regulated proteins has been studied in 19 primary phyllodes tumours, from patients with known follow-up, using immunohistochemical and molecular biological techniques. Overexpression of the p53 protein was observed in four cases and mutation in two cases. In only one case, the sequence alteration, at codon 273, was associated with overexpression of p53 protein and with strong expression of Ki-67 (30 per cent). This alteration was found in the primary, the recurrent, and the metastatic tumour samples. Moreover, the same p53 gene mutation, Arg273Cys, was detected in all tumour samples. No mutation was found in adjacent normal breast tissue, indicating that this was an acquired mutation. Unexpectedly, strong BAX expression was observed in the primary tumour. The patient died during the follow-up period. It is concluded that p53 gene status and an accumulation of BAX, both involved in the same apoptosis-controlling pathway, may be of prognostic relevance in phyllodes tumours.     

Phyllodes tumor of the breast: EGFR family expression and relation to clinicopathological features

The expression of EGFR family members was examined by immunohistochemistry in 22 phyllodes tumors, and the results were evaluated together with immunohistochemical findings for proliferation markers Ki67 and BM28, and the tumor suppressor gene product p53. Light and electron microscopy were performed in all cases. Clinical information was obtained from the medial records. We did find that expression of EG FR, c-erbB-3 and c-erbB-4 proteins could be detected in the neoplastic mesenchymal cells, and that the expression increased with increasing malignancy. Increased expressions of Ki67, BM28, p53 and EGFR family members in neoplastic cells were associated with malignancy and unfavorable clinical course. Furthermore, the expression of ER-alpha and PR in the epithelial cells of phyllodes tumors was increased compared to that in normal breast epithelium. Finally, the application of electron microscopy helped to identify a group of malignant tumors, revealing neoplastic cells with characteristic nuclear indentations, as well as an increasing number of myofibroblasts.     

Patterns of p53 expression in phyllodes tumors of the breast--an immunohistochemical study.

Authors performed an immunohistochemical analysis using monoclonal antibody to p53 protein on 15 cases of benign and malignant phyllodes tumor of the breast along with a review of other conventional clinicopathological parameters to investigate the meaning of p53 expression. The cases were composed of 8 benign and 7 malignant lesions. The pattern of p53 expression showed a statistically significant difference between these benign and malignant lesions (p < 0.005). None of the benign cases expressed p53 whereas 6 out of 7 malignant cases did. Among malignant phyllodes tumors, the pattern of expression was diffuse and strong in two cases while granular and relatively weak in the remaining 4 cases. p53 expression seemed to be a unique feature of malignant phyllodes tumors, thereby, one of the most significant parameters for the differentiation of benign and malignant phyllodes tumors of the breast.     

Balanced t(11;15)(q23;q15) in a TP53+/+ breast cancer patient from a Li-Fraumeni syndrome family

Li-Fraumeni Syndrome (LFS) is characterized by early-onset carcinogenesis involving multiple tumor types and shows autosomal dominant inheritance. Approximately 70% of LFS cases are due to germline mutations in the TP53 gene on chromosome 17p13.1. Mutations have also been found in the CHEK2 gene on chromosome 22q11, and others have been mapped to chromosome 11q23. While characterizing an LFS family with a documented defect in TP53, we found one family member who developed bilateral breast cancer at age 37 yet was homozygous for wild-type TP53. Her mother also developed early-onset primary bilateral breast cancer, and a sister had unilateral breast cancer and a soft tissue sarcoma. Cytogenetic analysis using fluorescence in situ hybridization of a primary skin fibroblast cell line revealed that the patient had a novel balanced reciprocal translocation between the long arms of chromosomes 11 and 15: t(11;15)(q23;q15). This translocation was not present in a primary skin fibroblast cell line from a brother with neuroblastoma, who was heterozygous for the TP53 mutation. There was no evidence of acute lymphoblastic leukemia in either the patient or her mother, although a nephew did develop leukemia and died in childhood. These data may implicate the region at breakpoint 11q23 and/or 15q15 as playing a significant role in predisposition to breast cancer development.     

p53 protein expression and gene mutation in phyllodes tumors of the breast

The malignant potential of mammary phyllodes tumors is difficult to assess on initial pathologic examination. Studies on the p53 tumor suppressor gene have shown that it has an important role in the development of a variety of malignancies, yet the specific contribution to the pathogenesis and development of the malignant potential of phyllodes tumor is largely unknown. We studied p53 protein expression in 25 cases of phyllodes tumors histologically classified as either malignant (12 cases) or benign (13 cases). Using microdissection approach, we also analyzed the p53 gene sequence in a case that demonstrated progression from benign to malignant phenotype. Nuclear p53 staining was detected in various proportions (1-90%) of neoplastic stromal cells of malignant tumors. No staining was found in benign tumors. Progression from benign to malignant phenotype was associated with a significant increase in the accumulation of p53 (more than 20 times). This was caused by an underlying missense mutation in exon 7, resulting in a change from Arg248 to Trp248 in the malignant component of the tumor. Stromal p53 over-expression was observed only in neoplasms histologically classified as malignant and was associated with an increased proliferation index (MIB-1 staining). These two markers may be used as useful adjuncts in the diagnosis of malignancy in difficult cases or when only a limited sample size is available. Somatic mutation in exon 7 of p53 gene in malignant phyllodes tumor points toward the importance of p53 in the malignant transformation of phyllodes tumors.     

Expression of the breast differentiation antigen NY-BR-1 in a phyllodes tumor of the vulva

We describe a phyllodes tumor of borderline malignancy in the labium majus of a 49-year-old woman. The histogenetic origin of phyllodes tumors in the vulva is controversial. Strong immunoreactivity for NY-BR-1, a novel breast differentiation antigen, was demonstrated within the epithelial components of the phyllodes tumor. A similar expression pattern was observed in mammary-like glands of the vulva. These findings provide further evidence that phyllodes tumors of the vulva might derive from mammary-like glands in the labium majus or from ectopic breast tissue.     

"Missed" diagnoses of phyllodes tumor on breast biopsy: pathologic clues to its recognition

Fibroadenoma and phyllodes tumors of the breast exhibit a continuum of pathologic features. We examined phyllodes tumors initially called fibroadenoma for features that may accurately classify the tumor as phyllodes tumor on the first biopsy specimen. The phyllodes tumors initially called fibroadenoma for features that may accurately classify the tumor as phyllodes tumors on the first biopsy specimen are examined. Fifteen patients with phyllodes tumors were studied, initially called FA or another term short of PT. These tumors were compared with 16 true fibroadenomas, all with needle-core biopsy followed by excision. Resected phyllodes tumors were larger on average than fibroadenoma, 6.8 cm (range = 1.7-16.2 cm) versus 2.6 cm (range = 1.0-4.8 cm). In needle-core biopsy cases, sampling was limited, even in large breast masses. p53 and cleaved caspase-3 were noncontributory. Ki-67 showed higher proliferation indices in phyllodes tumors versus fibroadenoma (4.8% vs 0.6%). Features suggesting phyllodes tumors include tissue fragmentation, increased stromal cellularity especially around glands, stromal overgrowth, and increased mitoses. Increased sampling of a large tumor will likely yield more correct diagnoses.     

The first case of Li-Fraumeni syndrome in Bosnia and Herzegovina: case report

Li-Fraumeni syndrome (LFS) is a very rare autosomal dominant and highly penetrant cancer syndrome characterized by early-onset primary tumours, including soft tissue and bone sarcoma, breast cancer, leukemia, brain tumours and adrenocortical carcinoma. Here we report the first evidence-based case of LFS in Bosnia and Herzegovina and the whole Balkan region. A ten year-old girl developed multiple primary tumours (rhabdomyosarcoma) during a period of eight years, as well as fibroadenoma of the breast. Sequential analysis revealed a germ line mutation of TP53 in exon 8, a common mutation in patients with LFS, in both the patient and her mother.     

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.     

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.     

Three germline mutations in the TP53 gene

Three germline mutations in the TP53 tumor-suppressor gene are reported, two of which are not reported previously. A missense mutation at codon 265 of TP53 was found in three patients of a family that complied with the definition of the Li-Fraumeni syndrome. A nonsense mutation in codon 306 was found in a woman who had had a rhabdomyosarcoma at age 4 and a subsequent breast cancer at age 22. She was part of a Li-Fraumeni-like family, but the parental origin of the mutation could not be traced. Finally, while screening for somatic alterations in TP53 in a series of 141 sporadic breast tumors, we detected a constitutional missense mutation in codon 235 in a woman diagnosed with breast cancer at age 26 and a recurrence 4 years later. The recurrence, but not the primary tumor, showed an additional missense mutation at codon 245 as well as loss of the wild-type allele. This suggests that the 245 mutation was particularly important for tumor progression and that there might exist heterogeneity in terms of cancer predisposition potential among the various germline TP53 mutations. Hum Mutat 9:157–163, 1997. © 1997 Wiley-Liss, Inc.     

Malignant phyllodes tumor of the breast with expression of osteonectin and vinculin

Phyllodes tumor is a very rare neoplasm which accounts for 2.5% of all fibroepithelial lesions of the breast. The mesenchymal component of a malignant phyllodes tumor frequently contains heterologous components. We report a case of malignant phyllodes tumor. The patient was a 40-year-old woman with a lump on the left breast. Histological examination revealed the lump to be a malignant phyllodes tumor with foci of liposarcomatous differentiation. The mesenchymal tumor cells, including those in the liposarcomatous components, were found to express vimentin, osteonectin and vinculin. However, they showed no immunoreaction to CAM 5.2, desmin, alpha-smooth muscle actin (ASMA), neuron-specific enolase (NSE) nor S-100. Ultrastructurally, the mesenchymal tumor cells were found to have abundant cytoplasmic organelles, but there was no evidence showing their differentiation to myofibroblasts. Further studies will be necessary to elucidate the significance of vinculin and osteonectin expression in malignant phyllodes tumor.     

TP53 and MYC gene alterations independently predict poor prognosis in breast cancer patients

We intended to establish the frequency of exon-specific TP53 gene alterations and the relation to patient and tumor characteristics and clinical outcome of patients with breast cancer. By using polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) and sequencing techniques, TP53 gene alterations were found in 59 (32%) of the 187 samples studied. Most of the TP53 changes (37%) were observed in exon 7. In patients with known follow up (median, 107 months), there was no significant association of the frequency of TP53 mutation with menopausal or nodal status, tumor size, or progesterone receptor status. TP53 gene alterations were more frequently present in estrogen receptor (ER)-negative (ER⁻) tumors (P = 0.04) and in tumors with an amplified HER2/NEU oncogene (P = 0.03). Univariate analysis showed that patients with a mutated TP53 in their primary tumors had shorter relapse-free (P = 0.01) and overall (P = 0.03) survival. Patients with a TP53 gene mutation in exon 8 may be identified as having a particularly rapid rate of relapse. In Cox multivariate regression analysis, which included age, menopausal status, lymph node status, tumor size, steroid-hormone-receptor status, and oncogene amplifications, both TP53 gene alteration and MYC amplification independently predicted poor prognosis, with relative hazard rates for TP53 and MYC of 1.8 and 1.6, respectively, in analysis for relapse-free survival and of 1.7 and 1.6, respectively, in analysis for overall survival. Genes Chromosom Cancer 16:170–179 (1996). © 1996 Wiley-Liss, Inc.     

TP53 and breast cancer

The TP53 gene (p53) is found altered in breast carcinomas in approximately 20-40% of all cases depending on tumor size and stage of the disease. It seems to be an early event in breast tumorigenesis. Several polymorphisms in the TP53 gene have been detected and their possible roles in breast cancer risk and association to type of cancer developed are discussed. The different mutation spectra seen in geographical and ethnic populations may be used to identify environmental exposure contributing to breast cancer development. The role of TP53 mutation as a prognostic marker is reviewed as well as its role as a predictor for therapy response. All data available on TP53 mutation analyses of human breast carcinomas, as well data from transgenic animal studies and experimental cell studies, support an important role for TP53 in mammary carcinogenesis.     

Discordance of genetic alterations between primary head and neck tumors and corresponding metastases associated with mutational status of the TP53 gene.

Ample molecular data are available on the progression from normal mucosa to invasive head and neck squamous cell carcinoma (HNSCC), but information on further genetic progression to metastatic disease is scarce. To obtain insight into the metastatic process, we compared 23 primary HNSCCs with 25 corresponding lymph node metastases (LNMs) and 10 corresponding distant metastases (DMs) with respect to TP53 mutations and patterns of loss of heterozygosity (LOH) based on 26 microsatellite markers on six chromosome arms (3p, 9p, 17p, 13q, 8p, and 18q). In 18 of the 23 patients, a TP53 mutation was detected in the primary tumor, and in all cases the same TP53 mutation was present in the corresponding LNM or DM. In nine of 20 patients with LNMs and three of seven patients with DMs, the LOH pattern of metastasis differed from that of the corresponding primary tumor by at least one marker. Microsatellite markers located on chromosome arms 13q, 8p, and 18q were most frequently discordant, providing evidence that alterations at these chromosomes occur late in HNSCC carcinogenesis. Moreover, evidence was found that DMs had developed directly from the primary tumor and not from LNMs. Remarkably, we observed that the mutational status of the TP53 gene is associated significantly with the degree of genetic differences between primary HNSCCs and corresponding metastases. All patients with TP53 wild-type primary tumors showed significantly more discordant LOH patterns in the corresponding LNMs and DMs than patients with TP53-mutated tumors. The percentages were 100% versus 27% (LNMs) and 100% versus 0% (DMs), respectively (P = 0.008 and P = 0.029; two-sided Fisher exact test). This finding suggests that TP53-mutated tumors need fewer additional genetic alterations to develop metastases compared with TP53 wild-type primary tumors.     

Evaluation of the molecular mechanisms involved in the gain of function of a Li-Fraumeni TP53 mutation

The TP53 tumor suppressor gene is the most frequent target for genetic alterations in human cancer. TP53 gene alterations may result in the gain of oncogenic functions such as neoangiogenesis and resistance to therapy. The TP53 germ line mutation c.659A>C (p.Y220S) was identified in stored DNA from related patients with Li-Fraumeni syndrome (LFS) who died after developing clinically aggressive tumors. All of the patients were treated with protocols that included doxorubicin hydrochloride (DX) as a pivotal drug. To define the in vitro mutational phenotype of this germ line mutation, we used murine fibroblasts explanted from wild-type (wt) and p53 knockout (KO) mice from the same littermate. p53Y220S and p53R175H fibroblasts, obtained from p53KO fibroblasts transfected with expression vectors encoding the human Y220S and R175H p53 mutants, respectively, exhibited resistance to DX treatment. Moreover, p53Y220S fibroblasts exhibited angiogenetic properties, and after DX treatment, p53Y220S failed to translocate into the nucleus and showed an increase in its cytosolic levels. DX treatment does not influence p53 distribution within the nuclear and cytosolic compartments in p53R175H fibroblasts. Peroxiredoxin II (Prx II), a protein that is involved in eliminating reactive oxygen species (ROS), showed increased expression intensity in p53Y220S fibroblasts after DX treatment, as observed by two-dimensional electrophoresis analysis. Moreover, Thioredoxin (Trx), a protein that cooperates with Prx II, is overexpressed in p53Y220S mutants under basal conditions. These data suggest a relationship between the presence of the p53Y220S mutation and enhanced levels of Prx II and Trx in mutant fibroblasts. Since one of the mechanisms of the DX antitumor effect has been ascribed to production of ROS, future studies will evaluate the involvement of PrxII and Trx in the chemoresistance of p53Y220S fibroblasts to DX.     

Hereditary TP53 codon 292 and somatic P16INK4A codon 94 mutations in a Li-Fraumeni syndrome family.

Li-Fraumeni syndrome is an autosomal dominant disorder that is characterized by various types of cancer in childhood and adult cases. Although hereditary TP53 mutation is very rare in different human cancers, it has been frequently reported in Li-Fraumeni syndrome. On the other hand, hereditary mutations of TP57KIP2, P15INK4B, and P16INK4A, which affect the cell cycle similar to TP53, were observed in some types of cancer. In a Turkish family with the diagnosis of Li-Fraumeni syndrome, we analyzed the mutation pattern of TP53, P57KIP2, P15INK4B, and P16INK4A in the peripheral blood, and loss of heterozygosity (homo/hemizygous deletion) pattern of TP53 and P15INK4B/P16INK4A in two tumor tissues. The propositus had a seminoma, his daughter a medulloblastoma, and one of his healthy cousins, a TP53 codon 292 missense point mutation (AAA-->ATA; Lys-->Ile) in the peripheral blood cells. Tumor tissue obtained from the propositus with the seminoma revealed loss of heterozygosity in the TP53 gene. In the analyses of tumor tissues from the propositus and his daughter, a P16INK4A codon 94 missense point mutation (GCG-->GAG; Ala-->Glu) was observed with the hereditary TP53 mutation. P16INK4A codon 94 mutation observed in our family is a novel mutation in Li-Fraumeni syndrome. No other gene alteration in TP53, P57KIP2, P15INK4B, and P16INK4A was observed. Existence of the P16INK4A mutation and the hereditary TP53 mutation with or without loss of heterozygosity in the TP53 gene (seminoma/medulloblastoma) may be evidence for a common mechanism involved in tumorogenesis. The gene alterations in TP53 and P16INK4A genes may be used as tumor markers in our family.