Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas.

BACKGROUND: Malignant gliomas are the most prevalent type of primary brain tumours but the therapeutic armamentarium for these tumours is limited. Platelet-derived growth factor (PDGF) signalling has been shown to be a key regulator of glioma development. Clinical trials evaluating the efficacy of anti-PDGFRA therapies on gliomas are ongoing. In this study, we intended to analyse the expression of PDGFA and its receptor PDGFRA, as well as the underlying genetic (mutations and amplification) mechanisms driving their expression in a large series of human gliomas. METHODS: PDGFA and PDGFRA expression was evaluated by immunohistochemistry in a series of 160 gliomas of distinct World Health Organization (WHO) malignancy grade. PDGFRA-activating gene mutations (exons 12, 18 and 23) were assessed in a subset of 86 cases by PCR-single-strand conformational polymorphism (PCR-SSCP), followed by direct sequencing. PDGFRA gene amplification analysis was performed in 57 cases by quantitative real-time PCR (QPCR) and further validated in a subset of cases by chromogenic in situ hybridisation (CISH) and microarray-based comparative genomic hybridisation (aCGH). RESULTS: PDGFA and PDGFRA expression was found in 81.2% (130 out of 160) and 29.6% (48 out of 160) of gliomas, respectively. Its expression was significantly correlated with histological type of the tumours; however, no significant association between the expression of the ligand and its receptor was observed. The absence of PDGFA expression was significantly associated with the age of patients and with poor prognosis. Although PDGFRA gene-activating mutations were not found, PDGFRA gene amplification was observed in 21.1% (12 out of 57) of gliomas. No association was found between the presence of PDGFRA gene amplification and expression, excepting for grade II diffuse astrocytomas. CONCLUSION: The concurrent expression of PDGFA and PDGFRA in different subtypes of gliomas, reinforce the recognised significance of this signalling pathway in gliomas. PDGFRA gene amplification rather than gene mutation may be the underlying genetic mechanism driving PDGFRA overexpression in a portion of gliomas. Taken together, our results could provide in the future a molecular basis for PDGFRA-targeted therapies in gliomas.

Breast carcinomas that co-express E- and P-cadherin are associated with p120-catenin cytoplasmic localisation and poor patient survival.

BACKGROUND: Changes in junctional catenin expression may compromise cadherin-mediated adhesion, increasing cell malignant properties such as invasive and metastatic abilities. Altered expression of alpha-, beta-, gamma- and p120-catenin has been reported to be associated with E-cadherin loss or decreased expression, in both breast carcinomas and breast cancer cell lines. AIMS AND METHODS: To investigate the expression and subcellular localisation of p120- and beta-catenin in a series of human invasive breast carcinomas, and correlate it with biological markers and clinicopathological parameters. RESULTS: Both catenins frequently exhibited a reduced membranous or cytoplasmic staining pattern. These alterations were significantly correlated with lack of both E-cadherin and oestrogen receptor-alpha expression. It was possible to associate the expression of beta-catenin with histological grade, tumour size and nodal status, suggesting a relevant role for this catenin as a prognostic factor. The majority of E- and P-cadherin co-expressing tumours were related to cytoplasmic expression of p120-catenin; in this group of breast carcinomas, patient survival was poor. CONCLUSION: Results indicate that p120-catenin cytoplasmic accumulation may play an important role in mediating the oncogenic effects derived from P-cadherin aberrant expression, including enhanced motility and invasion, particularly in tumours which maintain E-cadherin expression.

Mutations in gastrointestinal stromal tumors diagnosed by endoscopic ultrasound-guided fine needle aspiration.

Gastrointestinal stromal tumors (GISTs) are characterized by overexpression and mutations of c-Kit. Approximately 80% of c-Kit mutations occur in exon 11, being a response factor to imatinib (Gleevec) therapy. We aimed to assess whether c-Kit and PDGFRA mutation analysis of GISTs obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) could be routinely performed. Mutation analysis of c-Kit hotspot exons (9, 11, 13 and 17) and PDGFRA hotspot exons (12 and 18) was performed in aspirates 51 mesenchymal tumors. We identified c-Kit mutations in 61% of GIST cases, in accordance with previously published ranges (30-90%). Nearly 95% (19/20) of c-kit-mutant tumors carried exon 11 mutations. Mutation analysis is possible in FNA cell blocks and can assist in the diagnosis and therapeutic decisions in GIST cases.

Metaplastic breast carcinomas are basal-like tumours.

AIMS: Recently, an immunohistochemical panel comprising antibodies against HER2, oestrogen receptor (ER), epidermal growth factor receptor (EGFR) and cytokeratin (CK) 5/6 was reported to identify basal-like breast carcinomas, as defined by cDNA microarrays. Our aim was to analyse a series of metaplastic breast carcinomas (MBCs) using this panel plus two other basal markers (CK14 and p63) and progesterone receptor (PR), to define how frequently MBCs show a basal-like immunophenotype. METHODS AND RESULTS: Sixty-five cases were retrieved from the pathology archives of the authors' institutions and reviewed by three of the authors. Immunohistochemistry with antibodies for HER2, ER, EGFR, CK5/6, CK14 and p63 was performed according to standard methods. All but six cases (91%) showed the typical immunoprofile of basal-like tumours (ER- and HER2-, EGFR+ and/or CK5/6+). When CK14 and p63 were added to the panel, two additional cases could be classified as basal-like. The majority of MBCs lacked PR, except 4/19 (21%) carcinomas with squamous metaplasia. CONCLUSIONS: Our results demonstrate that MBCs show a basal-like phenotype, regardless of the type of metaplastic elements. Moreover, as these neoplasms frequently overexpress EGFR (57%), patients with MBC may benefit from treatment with anti-EGFR drugs.

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas.

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one-third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray-based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SEC61G. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2.

P-cadherin expression in glandular lesions of the uterine cervix detected by liquid-based cytology.

OBJECTIVE: To study P-cadherin aberrant expression as a possible marker for cervical adenocarcinomas in cytological samples. METHODS: We studied P-cadherin immunoexpression in liquid-based cervical cytology samples of biopsy-proven cervical lesions. RESULTS: We found a statistically significant correlation between P-cadherin expression and a cytological diagnosis of malignancy, either glandular or squamous (P < 0.0001). Twenty-two of 33 malignant cases showed P-cadherin membrane staining. None of the 30 benign cases tested showed membrane staining, but three of them displayed an aberrant nuclear P-cadherin expression. CONCLUSIONS: We concluded that P-cadherin can be used to discriminate between malignant and benign cervical cytological specimens, but not to discriminate glandular from squamous lesions.

c-KIT and PDGFRA in breast phyllodes tumours: overexpression without mutations?

AIM: To study the immunoexpression and mutational status of c-KIT and PDGFRA in a series of benign and malignant phyllodes tumours of the breast. MATERIAL/METHODS: Nineteen phyllodes tumours (13 benign and six malignant) were analysed by immunohistochemistry for the expression of c-KIT and PDGFRA. Direct sequencing of exons 9, 11, 13, and 17 of the c-KIT gene and exons 12 and 18 of PDGFRA was performed to check the mutational status of these two genes. RESULTS: c-KIT expression was found in 12 of the 19 cases (six of the 13 benign cases and all six malignant ones) and PDGFRA expression was seen in two of the 19 cases (one benign and one malignant case); the 2415 C>T alteration in exon 17 of the c-KIT gene was found in two cases (both benign); the intronic insertion IVS17-50insT and the 2866 G>T alteration in the coding region of exon 18 of the PDGFRA gene were also found in two cases (one malignant and one benign). However, the activating mutations described for these genes in gastrointestinal stromal tumours were not present. CONCLUSION: c-KIT expression is a frequent finding in phyllodes tumours, particularly in malignant cases; however, no activating mutations similar to those described for gastrointestinal stromal tumours were found. The PDGFRA does not seem to be an alternative pathway to tumour development in phyllodes tumours because neither expression nor activating mutations were noteworthy.

p63 Staining of myoepithelial cells in breast fine needle aspirates: a study of its role in differentiating in situ from invasive ductal carcinomas of the breast.

AIMS: One of the limitations of fine needle aspiration biopsy (FNAB) of the breast is in distinguishing invasive carcinoma (IDC) from ductal carcinoma in situ (DCIS). It has been proposed that the presence of myoepithelial cells overlying epithelial malignant cell clusters suggests DCIS. However, the recognition of myoepithelial cells in aspirates may be difficult. The aim of this study was to investigate a new nuclear myoepithelial cell marker, p63, a p53 homologue nuclear transcription factor, in a series of breast FNABs in an attempt to distinguish IDC from DCIS. METHODS: Papanicolaou stained smears from eight cases of pure DCIS and 15 cases of pure IDC with a histologically confirmed diagnosis were submitted to immunocytochemical analysis using the antibody 4A4 against p63. Two pathologists evaluated the presence of p63 positive cells overlying malignant cell clusters and admixed with malignant cells. The frequency of p63 positive cells in DCIS and IDC was compared using Fisher's exact test. RESULTS: p63 consistently stained the nuclei of myoepithelial cells, either overlying malignant cell clusters and/or admixed with malignant cells. p63 positive myoepithelial cells were seen in all DCIS cases and in nine of the 15 cases of IDC (p = 0.0375). In eight cases (three DCIS and five IDC), scattered p63+ epithelial malignant cells were seen. CONCLUSIONS: Although p63 positive myoepithelial cells are found more frequently in DCIS cases, their presence cannot be used as a criterion to rule out invasion in breast FNABs because they are present in up to 60% of invasive cases.

CD99/MIC-2 surface protein expression in breast carcinomas.

AIM: To evaluate the expression of CD99/MIC-2 surface protein in invasive breast carcinomas and demonstrate whether or not there is a relationship with tumour phenotype. METHODS AND RESULTS: Thirty-five invasive breast carcinomas, including five metaplastic carcinomas, were stained with CD99 primary antibodies using standard protocols based on streptavidin-biotin-peroxidase method. Four out of five metaplastic carcinomas expressed CD99/MIC-2 protein, three of them were matrix-producing carcinomas. From the other 30 cases, only an invasive apocrine carcinoma was positive. There was no statistical correlation between CD99 expression and the parameters analysed (histological typing and grading, proliferative index and nodal status). CONCLUSIONS: CD99/MIC-2 is expressed in breast carcinomas, especially in the matrix-producing variant of metaplastic carcinomas, which impairs its use as a marker to differentiate metaplastic carcinomas from primary and metastatic sarcomas of the breast. It seems to have no prognostic implications. However, phenotype similarities with other chondromyxoid tumours that also express the protein, like mesenchymal chondrosarcomas, suggest a relationship between MIC-2 reactivity and morphological differentiation.

Maspin expression in myoepithelial tumors of the breast.

Maspin is a mammary inhibitory serine protease that harbors tumor suppressor, tumor invasiveness-suppression and anti-angiogenic properties. It is consistently expressed by mammary myoepithelial cells. However, to the best of our knowledge, no assessment of maspin immunoexpression in myoepithelial cell lesions of the breast has been reported so far. We evaluated maspin expression by immunohistochemistry in five normal breast samples, one sclerosing papilloma (SP), one tubular adenomyoepithelioma (TA), one adenoid cystic carcinoma (ACC), one epithelial-myoepithelial carcinoma of the breast (EMC), and one malignant adenomyoepithelioma (MA). We also compared maspin expression with the expression of other classic myoepithelial markers in myoepithelial and secretory cells, as well as in stromal components of all samples. In normal breast samples, maspin expression was restricted to myoepithelial cell nuclei and cytoplasm. A strong nuclear and cytoplasmic maspin immunoreactivity was observed in the myoepithelial components of SP, TA, ACC, and EMC. In MA, maspin immunoreactivity was confined to the nucleus and cytoplasm of the cells lining tubular-like and papillary structures, as well as in squamous cells. The myoepithelial nature of maspin-positive cells was further confirmed by classic myoepithelial cell markers, including alpha-actin and S-100 protein. No stromal, neural or vascular components were immunostained by maspin. In spite of the small number of myoepithelial lesions here assessed, we suggest that maspin should be used in surgical pathology practice either as an additional marker in immunohistochemical panels defining a myoepithelial histogenesis in odd breast neoplasms, or in those cases in which the definite diagnosis relies on the myoepithelial cell layer identification.

Primary fibromatosis of the breast in a patient with multiple desmoid tumors--report of a case with evaluation of estrogen and progesterone receptors.

Fibromatosis (or desmoid tumor) is an infiltrative fibroblastic/myofibroblastic lesion presenting a moderate risk for local recurrence and no metastatic potential. Classically, these lesions are classified whether in abdominal or extra-abdominal sites, and may be multicentric or familial. Primary fibromatosis of the breast (PFB) is an uncommon lesion that shows histological similarities with abdominal fibromatosis (AF), and frequently poses difficulties in the differential diagnosis with other spindle cell tumors of the breast. It has been demonstrated that AF usually shows immunoreactivity for estrogen (ER) and progesterone (PR) receptors; conversely, in most of the studies, the cells from PFB are consistently negative for both receptors. We report on a case of a 41-year-old female with two desmoid tumors, affecting the abdominal wall and the breast tissue. To the best of our knowledge, there is no previous report in which hormonal receptors were evaluated in abdominal and mammary desmoid tumors in the same patient. We assessed the immunohistochemical expression of ER and PR in both lesions; while the AF showed immunoreactivity for both receptors, the cells from PFB were all negative. Although we have considered just this case, we still believe that these findings could support a distinctive etiopathogenesis of abdominal and mammary fibromatosis.