Pleomorphic adenoma and carcinoma ex pleomorphic adenoma: immunohistochemical demonstration of the association between tenascin expression and malignancy

AIMS: To investigate tenascin expression in salivary gland tumours. Tenascin is a matricellular protein that has been studied in several tumour types. Its expression has been correlated with tumour morphogenesis as well as with local invasiveness and tumour metastatic behaviour. METHODS AND RESULTS: The distribution pattern of tenascin in a series of 63 pleomorphic adenomas (PA) and 20 carcinomas ex- pleomorphic adenoma (Ca ex PA) was studied immunohistochemically. Ten normal adult salivary glands were used as controls. Tenascin surrounded the excretory ducts of normal adult salivary gland tissue. It was absent in the basement membrane compartment of both benign and malignant mixed tumours. In the interstitial compartment of the extracellular matrix, the fibro-hyaline type expressed tenascin in a statistically significantly (P < 0.001) lower number of PA cases (25%) in comparison with both malignant and benign areas of Ca ex PA (75% and 90%, respectively). In the Ca ex PA group, a statistically significantly difference (P < 0.001) was found in the frequency of tenascin deposits around aggregates of neoplastic cells between metastasizing (73%) and non-metastasizing neoplasms (0%). CONCLUSIONS: These findings strongly support the hypothesis that tenascin deposition is involved in the mechanisms of malignant transformation of pleomorphic adenomas into carcinomas as well as being associated with clinical disease progression.     

Lowp53 protein expression in salivary gland tumours compared with lung carcinomas

Summary Fifty-one salivary gland tumours (23 pleomorphic adenomas, 5 Warthin's tumours, 12 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 3 undifferentiated carcinomas and 1 acinic cell tumour) and 27 lung carcinomas (18 squamous cell carcinomas, 6 adenocarcinomas and 3 small cell carcinomas) were analysed immunohistochemically for the expression ofp53 nuclear phosphoprotein. Eight out of 51 (16%) salivary gland tumours werep53 positive. Three of these were benign and 5 malignant. All 3 benign salivary gland tumours were pleomorphic adenomas and expressed only occasional nuclear positivity with less than 1% of tumour cells positive. Of the 5p53-positive malignant tumours, 3 were mucoepidermoid carcinomas and 2 undifferentiated carcinomas. The malignant salivary gland tumours expressed more than 1% of positive nuclei in every case. Seventeen lung carcinomas werep53 positive (63%). Thirteen of these were squamous cell carcinomas, 3 were adenocarcinomas and 1 small cell lung carcinoma. The results show that mutations of thep53 gene may be infrequent in salivary gland tumours when compared with lung carcinomas. The relatively indolent course of some histological types of malignant salivary gland tumours could be associated with the preservation of the non-mutatedp53 gene in most of these tumours. The presence ofp53 positivity in some pleomorphic adenomas might, on one hand, suggest thatp53 gene alterations are also present in these tumours; on the other hand, the accumulation of thep53 protein in these tumours might also be due to some unknown mechanism, not necessarily related top53 gene mutation.     

New BAGE (B melanoma antigen) genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 have a cancer/testis expression profile

A first BAGE (B melanoma antigen) gene, BAGE1, was identified because it encodes a human tumour antigen recognised by a cytolytic T lymphocyte. Here, we characterised five new BAGE genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 and nine BAGE gene fragments mapping to the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. Genes and gene fragments share extensive regions of 90-99% nucleotide identity. We analysed the expression of BAGE genes on 215 tumours of various histological types and on nine normal tissues. Similar to BAGE1, the new BAGE genes are expressed in melanomas, bladder and lung carcinomas and in a few tumours of other histological types. All the normal tissues were negative, with the exception of testis. Our results show that human juxtacentromeric regions harbour genes, which are transcribed and translated, in addition to gene fragments that are generally not expressed. We suggest that the pattern of expression restricted to cancer/testis is a feature of the few genes mapping to juxtacentromeric regions.     

Epithelial membrane antigen and DOG1 expression in minor salivary gland tumours

Epithelial membrane antigen (EMA) and DOG1 are used as marker of epithelial cells, particularly the luminal cells, of salivary gland tumours. The aim of this study was to compare the EMA and DOG1 expression in tumours of minor salivary glands. Cases of pleomorphic adenoma (PA), basal cell adenoma (BCA), canalicular adenoma (CA), adenoid cystic carcinoma (ACC), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC) and epithelial-myoepithelial carcinoma (EMC) were submitted to immunohistochemistry for EMA and DOG1. In PA and BCA, EMA and DOG1 were observed in luminal cells, while in CA the tumour cells were negative for both proteins. The EMA and DOG1 pattern expression detected in EMC was similar to that one observed in benign tumours. In ACC, both myoepithelial e epithelial expressed EMA and DOG-1. PAC tumour cells were only positive for DOG1, whereas MEC were only positive for EMA. In conclusion, EMA and DOG1 expression in benign salivary gland tumours was similar to normal salivary gland tissue and can be used as good marker of tumoral cells derived from intercalated ducts or its progenitor cells, while in malignant salivary gland tumours EMA expression is, however, better used as an indicator of aggressive behavior than a marker of luminal cells.     

Association of hepatocyte growth factor expression with salivary gland tumor differentiation

To clarify the significance of hepatocyte growth factor (HGF) expression in salivary gland tumors, HGF distribution in tissue sections and HGF concentrations in saliva and serum were examined. Sixty salivary gland adenomas, 61 salivary gland carcinomas and three autopsy fetuses were studied. Hepatocyte growth factor expression was observed in the duct-type luminal cells by immunohistochemical staining and in situ hybridization. However, HGF failed to be expressed in acinar cells and myoepithelium of normal salivary gland tissue. Hepatocyte growth factor tended to be expressed more intensely in benign salivary gland tumors than in malignant salivary gland tumors (P < 0.0001). In highly malignant tumors, the expression was limited in some cases. Salivary and serological HGF concentrations of 18 patients, comprised of 12 benign cases and six malignant cases, were analyzed before and after operation by an ELISA system. The concentrations were distinctly elevated after operation, in both saliva and serum, compared to before operation (P < 0.0005). However, there were no significant relationships between HGF concentration and histology, age, gender, size or location. Our findings suggest that HGF may play an important role in the development of salivary ducts of normal salivary tissues and differentiation of ductal structures of their neoplasms, while HGF kinetics in saliva and serum would be less likely to reflect the neoplastic character, benign or malignant.     

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.     

Dendritic interstitial and myofibroblastic cells at the border of salivary gland tumors

OBJECTIVE: CD34-positive dendritic interstitial cells may be associated with the regulation of tumor growth. This association has been studied in various human neoplasms, especially skin tumors. In this study, we evaluated the distribution of dendritic interstitial cells and myofibroblastic cells at the tumor periphery of various benign and malignant salivary gland neoplasms. METHODS: Forty-nine cases of salivary gland tumors were selected: 16 pleomorphic adenomas, 12 Warthin tumors, 8 polymorphous low-grade tumors, 5 adenoid cystic carcinomas, 6 acinic cell carcinomas, and 2 mucoepidermoid carcinomas. Immunohistochemical analysis was performed by using antibodies for CD34 (dendritic cells) and alpha-smooth muscle actin (myofibroblast) on formalin-fixed, paraffin-embedded archival tissue. Staining intensity was graded as marked (3+), moderate (2+), weak (1+), and absent (0). RESULTS: Staining intensity for CD34 was 3+ in 24 (86%) of 28 benign tumors (pleomorphic adenomas and Warthin tumors) and 6 (29%) of 21 malignant tumors (polymorphous low-grade tumors, acinic cell carcinomas, adenoid cystic carcinomas, and mucoepidermoid carcinomas) and 2+ in 4 (19%) of 21 malignant tumors. None of the benign tumors displayed 2+ staining with CD34. Three (11%) of 28 benign and 11 (52%) of 21 of malignant tumors failed to stain with CD34. alpha-Smooth muscle actin staining was 3+ in 10 (36%) of 28 benign tumors and 6 (29%) of 21 malignant tumors, and 2+ in 11 (39%) of 28 benign and 2 (9%) of 21 malignant tumors. Five (18%) of 28 benign and 11 (52%) of 21 malignant tumors failed to stain with alpha-smooth muscle actin. CONCLUSION: We conclude that the dendritic interstitial cells and myofibroblastic cells may be associated with the regulation of tumor growth in salivary gland tumors.     

MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors.

The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.     

IMMUNOHISTOCHEMICAL STUDY OF OVEREXPRESSION OF FIBROBLAST GROWTH FACTOR-1 (FGF-1), FGF-2, AND FGF RECEPTOR-1 IN HUMAN MALIGNANT SALIVARY GLAND TUMOURS

Fibroblast growth factor-1 (FGF-1) and FGF-2 are broad spectrum mitogens. The expression of FGF-1, FGF-2, and their receptor, FGF receptor-1 (FGFR-1), was examined in malignant salivary gland tumours and normal salivary glands, using immunohistochemical methods. In seven cases of adenoid cystic carcinoma (ACC), both duct-like cells and modified myoepithelial cells were apparently immunopositive for FGF-1, FGF-2, and FGFR-1. In five cases of mucoepidermoid carcinoma (MC), all three types of tumour cells including epidermoid cells, mucous cells, and intermediate cells expressed immunoreactive FGF-1, FGF-2, and FGFR-1. In these malignant salivary gland tumours, increased expression of FGFR-1 correlated with the intensity of both FGF-1 and FGF-2 immunoreactivity. In contrast to malignant salivary gland tumours, eight cases of normal salivary gland showed negative immunostaining for FGF-1, FGF-2, and FGFR-1 while four cases were weakly immunoreactive for FGF and its receptor. These results demonstrate that malignant salivary gland tumours overexpress FGF-1, FGF-2, and FGFR-1 compared with normal salivary glands and suggest that these growth factors may play an important role in facilitating neoplastic progression in human salivary glands.     

Genetic analysis of the human oncoprotein MDM2 in benign and malignant tumors of the salivary gland.

INTRODUCTION: Genetic alterations of oncogene MDM2 promote malignant transformation of several human tumors. In tumors of the salivary gland, however, the genetic status of MDM2 has not been evaluated so far. METHODS AND RESULTS: Benign and malignant tumors of the salivary gland (6 pleomorphic adenomas, 3 Warthin's tumors, 1 adenocarcinoma, 1 basal cell adenocarcinoma, 1 mucoepidermoid carcinoma, 3 acinic cell carcinomas, 2 adenoid cystic carcinoma, 1 squamous cell carcinoma) were analyzed by fluorescence-based PCR techniques and immunochemistry for MDM2 gene amplification, MDM2 gene expression, MDM2 gene mutation, MDM2 RNA splicing and MDM2 accumulation. Data show that all samples contained nonamplified MDM2 genes with nonmutant zinc finger regions. However, in two benign and two malignant samples, novel MDM2 mRNA splicing variant types 1 and 2 were detected. Furthermore, three malignant tumors revealed significant nuclear MDM2 accumulation. Correlation between levels of MDM2 mRNA and MDM2 protein could not be detected in the specimens. CONCLUSION: The present study suggests that MDM2 gene mutation and gene amplification do not contribute to MDM2 accumulation detected in malignant tumors of the salivary gland. However, the role of novel MDM2 splicing variants in MDM2 expression and malignant transformation must be elucidated further.     

Expression of c-erbB-2 oncoprotein in salivary gland tumours: an immunohistochemical study.

Sections of formalin-fixed, paraffin-embedded primary salivary gland tumours were stained with a monoclonal antibody to the c-erbB-2 oncoprotein to determine the incidence and significance of expression of this protein. The series of 131 tumours comprised 33 cases of pleomorphic adenoma, 2 of malignant mixed tumour, 1 oxyphil adenoma, 31 Warthin's tumours, 4 basal cell adenomas, 6 mucoepidermoid carcinomas, 14 acinic cell carcinomas, 19 adenoid cystic carcinomas, 3 squamous carcinomas, and 18 poorly differentiated adenocarcinomas. Positive staining, as defined in previous studies, was present in five tumours (three cases of poorly differentiated adenocarcinoma, one mucoepidermoid carcinoma, and one adenoid cystic carcinoma). A review of the medical records of all patients did not disclose any clear difference between the clinical behaviour of positive and negative cases over a period of follow-up that ranged from 18 to 120 months. The findings of this study indicate that the protein product of the c-erbB-2 proto-oncogene is infrequently expressed in salivary gland tumours, and when it is localized on the tumour cell surface membrane, there is no clear evidence that this determines the biological behaviour of the tumour.     

Immunophenotypical profiles of salivary gland tumours: a new evidence for their histogenetic origin

The histogenetic origin of salivary gland tumours is not clear. In normal tissues smooth muscle actin (SMA) is expressed in myoepithelial cells, CK14 immunoreactivity is seen in myoepithelial and basal cells and CK10 in keratinized squamous epithelium. In this study, we examine the immunophenotypic properties of salivary gland tumours in order to obtain further insight into their histogenesis. 30 cases of salivary gland tumours (18 pleomorphic adenomas, 8 Warthin's tumours, 2 basal cell adenomas, 2 acinic cell carcinomas) were included in our study. Cytokeratin (CK) 10, CKI4, CKI7, CK18, CK 19, and smooth muscle actin (SMA) immunostains were applied to the sections. Immunoreactivities were detected and the statistical significance was evaluated by chi square test. SMA was not detected in Warthin's tumour (p < 0.0001). CK14 was found in all tumours except acinic cell carcinomas (p < 0.0001). CK10 immunoreactivity was observed in 5 Warthin's tumour. In conclusion, pleomorphic adenomas and basal cells adenomas originate from stem cells. Immunophenotypic profile of Warthin's tumour is suggestive of an embryological remnant origin.     

Differential expression of galectin-1 and galectin-3 in thyroid tumors. Potential diagnostic implications.

Carcinoma of the thyroid gland, the most frequently diagnosed endocrine malignancy, is often associated with early regional metastases. With the exception of papillary carcinoma, distinguishing benign from malignant thyroid neoplasms in the absence of metastatic disease is difficult. Recently, the vertebrate lectins galectin-1 and galectin-3 have been implicated in the regulation of cellular growth, differentiation, and malignant transformation of a variety of tissues. To determine whether these galectins have a role in thyroid neoplasia, we analyzed 32 specimens from thyroid malignancies (16 papillary, 7 follicular, 8 medullary carcinomas, and 1 metastasis to lymph node), 10 benign thyroid adenomas, 1 nodular goiter, and 33 specimens from adjacent normal thyroid tissue for the expression of galectin-1 and galectin-3 with immunohistochemical and immunoblotting techniques utilizing anti-galectin antibodies. All thyroid malignancies of epithelial origin (ie, papillary and follicular carcinomas) and a metastatic lymph node from a papillary carcinoma expressed high levels of both galectin-1 and galectin-3. The medullary thyroid carcinomas, which are of parafollicular C cell origin, showed a weaker and variable expression of these galectins. In contrast, neither benign thyroid adenomas nor adjacent normal thyroid tissue expressed galectin-1 or galectin-3. These results suggest that galectin-1 and galectin-3 may be associated with malignant transformation of thyroid epithelium and may potentially serve as markers for distinguishing benign thyroid adenomas from differentiated thyroid carcinomas.     

Expression of androgen,estrogen, and progesterone receptors in salivary gland tumors. Frequent expression of androgen receptor in a subset of malignant salivary gland tumors

The expression of sex hormone receptors in some tumors suggests a role for these receptors in tumor pathogenesis and therapy. Previous studies of the expression of estrogen and progesterone receptors in salivary gland tumors have reported conflicting results. We evaluated the immunohistochemical expression of androgen, estrogen, and progesterone receptors (AR, ER, and PR) in a series of 78 formalin-fixed, paraffin-embedded salivary gland tumors. Immunoreactivity for AR was seen in 14 of 14 carcinoma ex pleomorphic adenomas, 6 of 6 salivary duct carcinomas, and 2 of 2 basal cell adenocarcinomas but in only 2 of 10 acinic cell carcinomas, mucoepidermoid carcinomas, and adenoid cystic carcinomas each. AR expression was distributed evenly between the sexes. ER and PR were expressed in only a few cases of salivary gland tumors. All 26 benign salivary gland tumors were negative for AR, ER, and PR. The uniform expression of AR exclusively in a subset of malignant salivary gland tumors suggests a possible role for AR in the histogenesis and possibly in the clinical management of these malignant salivary gland tumors.