Pietro Leocata

Angiogenesis in intracranial meningiomas: immunohistochemical and molecular study

Much of the morbidity of intracranial meningiomas is related to the degree of tumour vascularity and the extent of peritumoural vasogenic oedema. Several studies have shown that vascular endothelial growth factor (VEGF) is up‐regulated in meningiomas, although its relationship with tumour vasculature is still unclear. In order to better understand the angiogenic assessment of intracranial meningiomas, we analysed its vascular pattern, both as number and as morphologic configuration of microvessels. Moreover, we investigated the mRNA‐VEGF expression, relating this expression to vascular pattern. A total of 40 intracranial meningiomas, classified as benign (31 cases), atypical (7 cases), and anaplastic (2 cases) were analysed. RT‐PCR analyses of mRNA‐VEGF and competitive‐PCR were performed. VEGF expression and microvessel density (MVD) were also immunohistochemically investigated. Grade II–III meningiomas showed numerous small microvessels (mean: 34), while the majority of Grade I showed few larger vessels (mean: 13.09) (P = 0.000003). A microvessel pattern overlapping into atypical subtype was found in eignt of the 31 (25.8%) Grade I meningiomas. A significant association was found between grading and vascular pattern (P = 0.0002), as well as between the MVD and the immunohistochemical expression of VEGF (P = 0.0005). The expression of mRNA agreed with the immunohistochemical expression of the protein (P < 0.0001). A total of 39 cases expressed the 121 VEGF isoform and, among these, 28 cases also expressed the 165 isoform. Only 9 cases expressed both isoforms 165 and 189. Grade II and III meningiomas showed a preponderant expression of soluble isoforms (121 and 165). These results prompt us to speculate that the microvessel pattern could underlie a higher metabolic demand, probably due to a rapid growth with a consequent worse clinical behaviour of the tumour. In this sense, the vascular pattern may be used as a prognostic factor, in order to mostly focus attention on those Grade I meningiomas which have a higher likelihood of either recurrence or development of perilesional oedema. The pattern of vasculature itself seems to be dependent on the types of VEGF isoforms: the Grade II–III meningiomas (that presented numerous microvessels) expressed the soluble isoforms 121 and 165, while the isoform 189 was more frequently detected in Grade I meningiomas.

CXCR4 expression correlates with the degree of tumor infiltration and BRAF status in papillary thyroid carcinomas.

Emerging evidence indicates that interactions between chemokine receptors and their ligands may have a critical role in several steps of tumor development, including tumor growth, progression, and metastasis. In this report, we retrospectively evaluated CXCR4 expression in a consecutive series of 200 papillary thyroid carcinomas. We investigated the relationship between the clinicopathological features of the tumors and mutations in the BRAF gene to verify whether overexpression of CXCR4 is linked to more aggressive behavior in thyroid tumors. CXCR4 protein expression was evaluated by immunohistochemical staining. A final staining score was calculated by adding the score representing the percentage of positive cells to the intensity score. The CXCR4 expression of each papillary thyroid carcinoma sample was normalized by calculating the z score for each final staining score. Univariate analysis was used to correlate CXCR4 expression with the papillary thyroid carcinoma variant, the degree of neoplastic infiltration, the American Joint Commission on Cancer stage, the presence of lymphocytic thyroiditis and the mutation status of the BRAF gene. Multiple regression analysis confirmed a strong association between CXCR4, BRAF mutation and the degree of neoplastic infiltration. These data clearly indicate that the chemokine receptor expression induced by oncogenic activation could be the major determinant of the local aggressiveness of neoplastic cells. In conclusion, our data indicate that CXCR4 expression and BRAF mutation status could cooperatively induce and promote a more aggressive phenotype in papillary thyroid carcinoma through several pathways and specifically increase the tumors’ spread outside of the thyroid gland.

Low prevalence of human papillomavirus infection in the healthy oral mucosa of a Northern Italian population

BACKGROUND Oral cancer is the sixth most common malignancy in developed countries, representing almost 3% of malignant tumors. Tobacco use and alcohol consumption are well-established risk factors. However, the observation that most patients with oral cancer have not been exposed to these risk factors suggests that additional causes may promote oral carcinogenesis. A link has been suggested between human papillomavirus (HPV) and oral cavity cancer but the significance of HPV contribution to oral carcinogenesis as well as the prevalence of HPV infection in normal oral cavity mucosa remains debated. METHODS In this study, the prevalence of oral HPV infection was evaluated in 81 randomly selected Northern Italian subjects with clinically normal oral mucosa using a nested PCR on DNA extracted by oral smears. RESULTS AND CONCLUSIONS No HPV-related lesions were detectable in any of the smears analyzed by cytological approach. nPCR identified HPV DNA in only one (1.2%) of the specimens obtained from clinically healthy oral mucosa and subsequent characterization assigned the positive case to HPV type 90. These data suggest that the incidence of HPV infection in the healthy population might be very low and that other risk factors are likely responsible to promote oral carcinogenesis.

CD34 microvessel density and VEGF expression in basal and squamous cell carcinoma.

Angiogenesis is a central process in the growth of solid tumors. The purpose of our study was to analyze the angiogenic pattern in squamous and basal cell carcinomas and to point out differences in microvessel density that could explain their different biological behaviour. Thirty-nine skin tumors (26 basal and 13 squamous cell carcinomas) were analyzed. In all samples, the microvessels density (MVD) and the levels of vascular endothelial growth factor mRNA (VEGFmRNA) were analyzed, together with the inter-relationship between these two variables. Using the median value of the entire series (33 vessels per 2.22 mm2), tumors with low and high MVD were identified. The majority of cancers with high vascularization belonged to the squamous histotype (12 of 39), while 19 of the 26 basal cell carcinomas showed a lower number of microvessels than the median value (p = 0.0001). The median value of VEGFcDNA quantitation allowed us to distinguish tumors with high VEGF expression (> 470 molecules cDNA) from those with low (< or = 470 molecules) VEGF expression: 20 of the 26 basal cell carcinomas showed low VEGF expression, while 11 of the 13 squamous cell carcinomas showed high VEGFcDNA levels (p = 0.0003). Moreover, a significant association between a high microvessel density and high VEGFmRNA levels (p = 0.006) was found. Furthermore, when studying VEGF expression by immunohistochemistry, we obtained similar results and noted a correlation with VEGFmRNA expression (p < 0.0001). The association between high vascularization, high VEGF levels, and squamous cell histotype suggests the possible role of neoangiogenesis in determining the more aggressive biological behaviour of this type of cancer.

Biological effects of low frequency high intensity ultrasound application on ex vivo human adipose tissue.

In the present work the effects of a new low frequency, high intensity ultrasound technology on human adipose tissue ex vivo were studied. In particular, we investigated the effects of both external and surgical ultrasound-irradiation (10 min) by evaluating, other than sample weight loss and fat release, also histological architecture alteration as well apoptosis induction. The influence of saline buffer tissue-infiltration on the effects of ultrasound irradiation was also examined. The results suggest that, in our experimental conditions, both transcutaneous and surgical ultrasound exposure caused a significant weight loss and fat release. This effect was more relevant when the ultrasound intensity was set at 100% (∼ 2.5 W/cm2 for external device; ∼19–21 W/cm2, for surgical device) compared to 70% (∼ 1.8 W/cm2 for external device; ∼13–14 W/cm2 for surgical device). Of note, the effectiveness of ultrasound was much higher when the tissue samples were previously infiltrated with saline buffer, in accordance with the knowledge that ultrasonic waves in aqueous solution better propagate with a consequently more efficient cavitation process. Moreover, the overall effects of ultrasound irradiation did not appear immediately after treatment but persisted over time, being significantly more relevant at 18 h from the end of ultrasound irradiation. Evaluation of histological characteristics of ultrasound-irradiated samples showed a clear alteration of adipose tissue architecture as well a prominent destruction of collagen fibers which were dependent on ultrasound intensity and most relevant in saline buffer-infiltrated samples. The structural changes of collagen bundles present between the lobules of fat cells were confirmed through scanning electron microscopy (SEM) which clearly demonstrated how ultrasound exposure induced a drastic reduction in the compactness of the adipose connective tissue and an irregular arrangement of the fibers with a consequent alteration in the spatial architecture. The analysis of the composition of lipids in the fat released from adipose tissue after ultrasound treatment with surgical device showed, in agreement with the level of adipocyte damage, a significant increase mainly of triglycerides and cholesterol. Finally, ultrasound exposure had been shown to induce apoptosis as shown by the appearance DNA fragmentation. Accordingly, ultrasound treatment led to down-modulation of procaspase-9 expression and an increased level of caspase-3 active form.

Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection.

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.

Cyclin D3 immunoreactivity in follicular lymphoma is independent of the t(6;14)(p21.1;q32.3) translocation or cyclin D3 gene amplification and is correlated with histologic grade and Ki‐67 labeling index

An abnormal expression of cyclin D3, a key regulator of the cell cycle, has been documented in a variety of human malignancies, and the cyclin D3 gene, mapping to 6p21, may be deregulated in plasma cell myeloma and non‐Hodgkins lymphoma as a result of the t(6;14)(p21.1;q32.3) translocation and/or gene amplification. In the current study, we for the first time investigated by immunohistochemistry and fluorescence in situ hybridization (FISH) the prevalence of cyclin D3 abnormalities in follicular lymphomas (FLs), comparing the results with traditional clinicopathologic characteristics, p27 and skp2 immunoreactivity (IR) and Ki‐67 labeling index (LI). Cyclin D3, skp2 and Ki‐67 IR significantly increased from grade I to grade III FL (p = 0.0003, p = 0.0001 and p < 0.0001, respectively), while p27 IR was significantly (p < 0.0001) more prevalent in low‐grade tumors. Cyclin D3 IR was directly correlated with the Ki‐67 LI (p < 0.0001) and inversely correlated with p27 IR (p = 0.050). None of the 13 cases analyzed by FISH showed the t(6;14) translocation, but in 2 (15.3%) grade I FLs 3 cyclin D3 signals were detected. Cohybridization with probes specific for the centromeric region and the long arm of the chromosome 6 indicated trisomy in one case and a pattern highly suggestive for the presence of an isochromosome 6p in the other case. Our data suggest that the t(6;14) translocation may be extremely uncommon in FL and that a deregulated expression of cyclin D3, possibly due to epigenetic mechanisms, may be involved in the pathogenesis of high‐grade tumors.

Solitary fibrous tumor of the oral cavity with a predominant leiomyomatous-like pattern: A potential diagnostic pitfall

The diagnosis of solitary fibrous tumor (SFT) is usually straightforward if the typical morphologic features, including a wide variety of growth patterns, are identified. We report the clinical, radiologic, and pathologic findings of a rare case of intraoral SFT which exhibited a predominant leiomyomatous-like appearance, closely reminiscent of a leiomyoma, at both incisional and excisional biopsy. Histologically, the tumor was composed predominantly of intersecting fascicles of eosinophilic spindle-shaped cells, variably set in a fibrous stroma. A focal hemangiopericytoma-like growth pattern with alternating hypercellular and hypocellular areas, as well as the deposition of dense keloid-type collagen, raising the suspicion of SFT, could be identified only after a careful examination of the whole tumor. Immunohistochemistry was helpful in confirming the diagnosis of SFT, revealing a diffuse staining of neoplastic cells for vimentin, CD34, bcl-2 protein, and, focally, CD99. Myogenic markers (alpha-smooth muscle actin, desmin, h-caldesmon) were not expressed. The pathologist should be aware of this variant of intraoral leiomyomatous-like SFT to avoid a misdiagnosis of leiomyoma. The distinction of SFT from leiomyoma in the oral cavity is important to assure both correct treatment and prognostic information.

Unicentric angiofollicular hyperplasia (Castleman's disease) of the parotid: A case report

Angiofollicular lymph node hyperplasia, or Castlemans disease, is a condition of uncertain cause usually presenting with mediastinal lymphadenopathy. Occasionally other lymph node groups may be involved with or without associated systemic manifestations. A case of Castlemans disease involving parotid lymph nodes is reported in a 16-year-old female patient who presented with a painless swelling that initially was noticed at the age of 3 years. Diagnosis was established by histopathologic examination of the respected specimen. Histologic and clinical features of Castlemans disease are also discussed.

Follicular-derived neoplasms: Morphometric and genetic differences

Background: The distinction between follicular adenomas (FAs) and well differentiated follicular and papillary carcinomas is often a demanding task and sometimes only intuitive. Aim: We report an histomorphological evaluation of follicular neoplasms [FAs, follicular carcinomas (FCs), and follicular variant of papillary carcinomas (FVPTCs)], supported by a qualitative and quantitative image analysis and by a molecular characterization. Material and methods: Tumor fibrosis and haemorrhage, neoplastic capsule thickness, follicle diameter, number of neoplastic cells, nuclear diameter of neoplastic cells, vessels density, vessels area and intratumoral distribution were evaluated. Ras and BRAF mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements were analyzed. Correlations with clinico-pathological features have been studied. Results: We found that FAs had a more extensive intratumoral haemorrhage, while malignant neoplasms were characterized by an evident fibrosis, higher cellularity and larger size. FVPTCs had higher nuclear diameter; cells count was higher in the minimally invasive follicular thyroid carcinomas, as well as a thickener neoplastic capsule. The CD34 stain showed a higher microvessel density in the FVPTCs group. A higher peripheral vessels distribution was observed only in malignant neoplasms. We observed overall Ras mutations in 2.4% of adenomas, in 41.5% of FVPTCs, and in 44.8% of FCs. It is outstanding that there is a marked difference in the Ras mutation distribution between the benign and malignant tumors in our series. Conclusions: We found that genotyping of Ras gene family together with an accurate analysis of selected morphological features could help in the differential diagnosis of follicular-derived thyroid neoplasms.