MAML2 rearrangement in Warthin's tumour: a fluorescent in situ hybridisation study of metaplastic variants.

BACKGROUND: Warthin's tumour (WT) is a common benign lesion of the major salivary glands. The nature of WT remains controversial, with particular regard to the presence of clonal chromosomal abnormalities, including the t(11;19) translocation involving the CRTC1 and MAML2 genes, that have been identified in both WT and mucoepidermoid carcinoma. In this study, we focused our attention on metaplastic WT variants, and we conducted a fluorescent in situ hybridisation (FISH) analysis for the presence of MAML2 gene rearrangement. METHODS: Dual-colour FISH analysis was performed on paraffin-embedded sections of eight WTs showing metaplastic changes (five with squamous metaplasia, two with mucinous metaplasia and one with both) using a MAML2 break-apart probe. RESULTS: Presence of split signals indicative of gene rearrangement was identified in a subset of cells in areas of squamous metaplasia in two samples of WT. No rearrangement was observed in the oncocytic epithelium, in lymphocytes and in areas of mucinous metaplasia. CONCLUSIONS: The presence of a small subpopulation of cells carrying MAML2 rearrangement in areas of squamous metaplasia within WT could predispose these lesions to malignant transformation in mucoepidermoid carcinoma and could represent a molecular link between the two entities.

Prognostic significance of the alterations of the G1-S checkpoint in localized leiomyosarcoma of the peripheral soft tissue.

BACKGROUND: The aim of this study was to investigate the expression of cell cycle regulators p53, p16, cyclin-D1, and retinoblastoma (Rb) gene protein in leiomyosarcoma of the peripheral soft in order to identify expression profiles potentially useful for clinical prognostic purposes. MATERIALS AND METHODS: A tissue microarray representing 70 localized leiomyosarcomas of the limbs and limb girdles was created with 3 representative cores from each tumor. Immunohistochemical staining was performed for p53, p16, cyclin-D1, and Rb using standard techniques. Staining was scored as either absent-low (<20% of neoplastic cells) or moderate-diffuse (≥20%). Outcome analysis was performed for local recurrence-free survival (LFS), metastatic disease-free survival (MDFS), and disease-specific survival (DSS). RESULTS: Kaplan-Meier analysis of survival revealed that no single alteration of the factors examined was associated with outcome, but tumors showing concomitant alteration of p16 and p53 were characterized by reduced MDFS and DSS (P = 0.01 and P < 0.001, respectively). In addition, patients who received adjuvant therapy consisting of radiotherapy alone or radiotherapy and chemotherapy had a better DSS than those receiving surgery alone or surgery and chemotherapy (P = 0.05). In multivariate analysis, altered p16/p53 remained the only parameter predictive of MDFS and DSS (P = 0.048, hazard ratio [HR] = 2.488, 95% confidence interval [95% CI] 1.006-5.116; P = 0.043, HR = 2.498, 95% CI 1.029-5.909, respectively). CONCLUSIONS: Accumulation of cell cycle alterations represents a prognostic indicator in localized soft tissue leiomyosarcoma, and in particular altered p16/p53 expression is associated with an unfavorable prognosis. This may help the clinical management of patients with leiomyosarcomas.

Immunohistochemical investigation of tumorigenic pathways in sinonasal intestinal-type adenocarcinoma. A tissue microarray analysis of 62 cases.

AIMS: Sinonasal intestinal-type adenocarcinoma (ITAC) is an uncommon neoplasm morphologically similar to colorectal adenocarcinoma, with a well-recognized association with occupational exposure to wood or leather dusts. Here, we analyse several gene products with pivotal roles in tumorigenesis, including p53, p16, deleted in colon cancer (DCC), retinoblastoma, adenomatous polyposis coli, ß-catenin, E-cadherin and CD10, and discuss their relation to clinical behaviour and to similar pathways in colorectal adenocarcinomas. METHODS AND RESULTS: Immunohistochemical analysis of 62 ITACs was conducted on a tissue microarray. Aberrant expression of p53 and p16 were the most commonly observed alterations (61.3% and 64.5% of cases, respectively). Analysis according to the histological subtype showed that p53 overexpression was less frequent in mucinous ITACs (35.3% versus 71.1%, P = 0.018), while loss of DCC and E-cadherin were observed more frequently in this subtype (76.5% versus 31.1%, P=0.002 and 82.4% versus 31.1%, P<0.001, respectively). No correlation was found between the aberrant expression of these and clinical behaviour while mucinous adenocarcinomas had a significantly worse prognosis, with shorter disease-free interval and overall survival (P=0.005 and P<0.001, respectively). CONCLUSIONS: Mucinous ITACs appear to follow a distinct molecular pathway(s) from the non-mucinous variants, and pursue an aggressive clinical behaviour.

Tumour suppressor gene TP53 mutations in atypical vascular lesions of breast skin following radiotherapy.

AIMS: Atypical vascular lesions (AVL) occurring at the site of radiotherapy represent an uncommon but well-documented complication in the setting of breast-conserving therapy for breast carcinoma. Although the biological behaviour of AVL has been regarded as benign, it has been suggested that AVL may represent a precursor of angiosarcoma. A better understanding of the biology of AVL is essential in order to assess appropriate patient management. The aim of the present study was to investigate alterations of tumour suppressor gene TP53 in a series of radiation-induced AVL and angiosarcomas (AS). METHODS AND RESULTS: Direct sequencing analysis of the TP53 gene showed the presence of at least one variation in 10 of 12 (83.3%) AVL and in seven of eight (87.5%) AS. The most common alteration in both categories was the P72R polymorphism in exon 4. One angiosarcoma sample carried a pathogenetically relevant disruptive mutation c.592delG, a frameshift deletion in exon 6, causing a premature stop codon. CONCLUSIONS: The presence of TP53 alterations suggests that its mutational inactivation may be implicated in the pathogenesis of radiation-associated vascular proliferations. The common mutational pathway suggested by our data supports the hypothesis that AVL and AS are biologically related entities, most probably representing the extremes of a morphological continuum.

RANK and RANK Ligand Expression in Parotid Gland Carcinomas.

Recently, it has been reported that deregulation of the receptor activator of NFkB ligand (RANKL)/RANK signaling axis results in salivary gland tumor development in a mouse transgenic model. The aim of this study was to ascertain RANKL and RANK protein expression in a series of primary parotid gland carcinomas and to correlate it with clinicopathologic parameters. Formalin-fixed paraffin-embedded tumor samples from 46 consecutive cases of parotid gland carcinoma were selected for this study. For comparison, we examined a group of 40 randomly chosen parotid gland adenomas, including 20 pleomorphic adenomas, 10 myoepitheliomas, and 10 Warthin tumors. Immunohistochemical analysis for RANK and RANKL was conducted on tissue microarrays. Overall, 33 carcinomas (71.7%) were scored as positive for RANK and 25 (54.3%) for RANKL. The expression of both RANK and RANKL was significantly higher in carcinomas than in adenomas as only 6 (15%) adenomas were positive for RANK, and RANKL was negative in all benign tumors (P<0.001 for both, Fisher exact test). Some histologic types, including salivary duct carcinoma, mucoepidermoid carcinoma, and carcinoma ex-pleomorphic adenoma presented a high frequency of RANK and RANKL expression. No significant correlation was observed between RANK/RANKL expression and clinical parameters. Our study indicates that the expression of RANK and RANKL in parotid gland neoplasms is associated with the acquisition of a malignant phenotype and this pathway may represent an attractive therapeutic target in patients with parotid gland carcinomas.

Expression of cyclooxygenase-2 in osteosarcoma of bone.

Several studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in human malignancies, where it produces high levels of prostaglandins and contributes to tumor growth. In this study we have analyzed the expression of COX-2 in a series of 48 skeletal osteosarcomas of different subtypes by immunohistochemistry. In addition, we examined the effects of the specific COX-2 inhibitor Celecoxib on the growth of the human osteosarcoma cell line SaOS-2. Immunoreactivity for COX-2 was observed in 39 out of 48 tumors (81.2%), 30 (76.9%) of which showed a moderate or diffuse immunostaining. Considering the group of 42 primary osteosarcomas, COX-2 immunoreactivity was significantly higher in high grade osteosarcomas, where moderate or diffuse expression was detected in 23 out of 32 cases (71.8%), than in low grade osteosarcomas, where moderate or diffuse expression was detected in 2 out of 10 cases (20%) (P = 0.008, Fisher exact test). In addition, low COX-2 expression was always associated with a good response to chemotherapy (5 out of 5 cases), whereas moderate or diffuse COX-2 expression was associated with a good response in 11 out of 20 cases (55%) (P = 0.12, Fisher exact test). In SaOS-2 osteosarcoma cells, which express COX-2, treatment with Celecoxib determined inhibition of cell proliferation and induction of apoptosis. These results indicate that COX-2 is expressed at high levels in high grade osteosarcomas and support the use of COX-2 inhibitors to improve both the tumor response to chemotherapy and the outcome of osteosarcoma patients.

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

Intestinal metaplasia of the sinonasal mucosa adjacent to intestinal-type adenocarcinoma. A morphologic, immunohistochemical, and molecular study.

It has been hypothesized that the development of sinonasal intestinal-type adenocarcinoma (ITAC) occurs through intestinal metaplasia (IM) of the respiratory and/or glandular epithelium. The aim of this study was to characterize the histological, immunohistochemical, and molecular features of sinonasal IM. Histologic slides from 29 consecutive surgical specimens of ITAC were retrieved. Sections were stained for CDX2, cytokeratin 20 (CK20), MUC2, and p53. The status of TP53 gene exons 4-9 was assessed separately in areas of IM and in ITAC. Foci of IM were detected in eight cases (27.5%). They were all positive for CK20 and CDX2, while MUC2 was detected in six cases (75%). In six cases (75%), the metaplastic foci showed signs of dysplasia, including nuclear enlargement with increased nucleus to cytoplasm ratio, nuclear hyperchromasia, loss of nuclear polarity, and presence of prominent nucleoli. P53 nuclear immunoreactivity was observed in four cases. TP53 gene sequencing was successfully performed in six cases and revealed the same mutation in both IM and ITAC in two cases (c.832C > T and c.215G > C), while another ITAC showed a mutation that was not present in the adjacent IM (c.536A > G). In conclusion, our study suggests a possible clonal relationship between areas of sinonasal IM and ITAC, indicating that IM may represent a precursor lesion of ITAC. Improving the knowledge on the morphological and molecular features of IM is a key step to identify reliable biomarkers to determine the risk of sinonasal ITAC development.

KIT genetic alterations in anorectal melanomas.

BACKGROUND: Mucosal melanomas (MM) represent a heterogeneous tumour population that exhibits site-specific molecular profiles. AIMS: In a multicentre retrospective study, we investigated KIT aberrations in primary anorectal (AR) melanomas compared with melanoma metastatic to the gastrointestinal (GI) tract. METHODS: Primary AR MM (n=31) and GI metastatic melanoma (n=27) were studied for KIT mutations on exons 11, 13, 17 and 18 by high-resolution melting analysis, direct sequencing and c-KIT expression by immunohistochemistry. Selected cases were also investigated for increased KIT gene copy number by fluorescent in situ hybridisation. RESULTS: Functional KIT mutations were demonstrated in 11/31 (35.5%) of AR melanomas and in 1/26 (3.8%) of GI melanoma metastases (p=0.004). A significant difference emerged between primary and metastatic MM with regards to KIT-positive immunostaining (p=0.002). Immunohistochemical c-KIT protein overexpression did not correlate with KIT mutational status. Increased KIT copy number was demonstrated in 5/20 AR primary cases. CONCLUSIONS: The rate of functional mutations in KIT is significantly higher in AR MM than in GI metastatic melanoma. KIT protein overexpression does not correlate with KIT mutations and cannot be used for screening purposes. Recognising the molecular heterogeneity of MM helps to identify patients who require a different therapeutic approach.

Rosiglitazone impairs proliferation of human adrenocortical cancer: preclinical study in a xenograft mouse model.

Adrenocortical carcinoma (ACC) is a rare aggressive tumor with a poor prognosis. The lack of a specific and effective medical treatment is due to the poor knowledge of the mechanisms underlying tumor growth. Research on potential drugs able to specifically interfere with tumor proliferation is essential to develop more efficacious therapies. We evaluated for the first time the in vivo effect of rosiglitazone (RGZ), an anti-diabetic drug with in vitro anti-tumor properties, on ACC proliferation in a xenograft model obtained by s.c. injection of human ACC H295R cells in athymic mice. When the tumor size reached 5 mm, animals were allocated to 5 mg/kg RGZ- or water-treated groups. Tumor volume was measured twice a week. A significant reduction of tumor growth in RGZ versus control (control) group was observed and was already maximal following 17 day treatment (1-T/C=75.4% (43.7-93.8%)). After 31 days of treatment, mice were killed and tumor analyzed. Tumor histological evaluation revealed characteristics of invasiveness, richness in small vessels and mitotic figures in control group, while RGZ group tumors presented non infiltrating borders, few vessels, and many apoptotic bodies. Tumor immunohistochemistry showed that Ki-67 was reduced in RGZ versus control group. Quantitative real-time RT-PCR demonstrated a significant reduction in the expression of angiogenic (VEGF), vascular (CD31), proliferation (BMI-1), and anti-apoptotic (Bcl-2) genes in RGZ versus control group tumors. The same inhibitory effects were confirmed in in vitro RGZ-treated H295R. Our findings support and expand the role of RGZ in controlling ACC proliferation and angiogenesis in vivo and in vitro.

Epidermal growth factor receptor expression and gene copy number in sinonasal intestinal type adenocarcinoma.

Sinonasal intestinal type adenocarcinoma (ITAC) is a rare subtype of adenocarcinoma strongly associated with professional exposure to wood or leather dusts. It is an aggressive tumor, with an estimated 5-year survival of 40%. Herein, we report a series of 55 cases tested for epidermal growth factor receptor protein expression and epidermal growth factor receptor gene copy gains, through immunohistochemistry and fluorescence in situ hybridization. Eighteen tumors (32.7%) showed EGFR positivity, seven of which exhibited high expression levels of the receptor. The frequency of EGF-R overexpression was significantly higher in tumors from woodworkers (6 of 14, 42.8%) than in tumors from leatherworkers (2 of 21, 9.5%), or arising in subjects with no known occupational history (0 of 8) (P=0.015, Pearson chi square). No correlation was found with other clinico-pathological parameters, including histologic subtype, stage, overall, and disease free survival. In cases with EGFR overexpression, fluorescent in situ hybridization analysis revealed disomy in three adenocarcinomas, chromosome 7 polysomy in two, and EGFR gene amplification in three. In conclusion, a subset of ITAC, mostly occurring in woodworkers, express high levels of EGFR and this is often associated with either gene amplification or chromosome 7 polysomy. EGFR targeted therapies could therefore be investigated prospectively in this group of tumors.