Anna Calzolari

Expression of transforming growth factor β isoforms in osteosarcoma variants: association of tgfβ1 with high-grade osteosarcomas

Studies on osteosarcoma cell lines point to the potential importance of transforming growth factor β (TGFβ) as an autocrine factor which controls the growth of human osteosarcomas. To define further the role of TGFβ isoforms in these neoplasms, a series of 27 osteosarcomas was studied using immunohistochemical, mRNA in situ hybridization, and reverse transcriptase‐polymerase chain reaction (RT‐PCR) techniques. All 14 central high‐grade osteosarcomas, two telangiectatic osteosarcomas, and one high‐grade surface osteosarcoma showed cytoplasmic immunoreactivity for TGFβ1, ‐2, and ‐3. The expression of TGFβ1 was moderate or diffuse in 14 cases (82·3 per cent), while low expression was detected in only three cases (17·7 per cent). For TGFβ2 and ‐3, only moderate or diffuse staining was observed. Low‐grade parosteal and periosteal osteosarcomas showed low or undetectable levels of TGFβ1, while TGFβ2 and ‐3 were moderately or diffusely expressed. Finally, three dedifferentiated parosteal osteosarcomas were diffusely positive for TGFβ1, ‐2, and ‐3 in the high‐grade component, while in the low‐grade component, available for analysis in two of these cases, TGFβ1 was demonstrated in a few neoplastic cells, and TGFβ2 and ‐3 maintained a diffuse distribution. Statistical analysis of these data showed that high‐grade osteosarcomas had a significantly higher expression of TGFβ1 than low‐grade osteosarcomas, while levels of TGFβ2 and ‐3 were comparable in the two groups (p<0·001; p=0·3; p=0·3, respectively; Fishers exact test). Similarly, mRNA levels of TGFβ1 detected by in situ hybridization were significantly higher (p=0·04, Fishers exact test) in high‐grade osteosarcoma variants, while no differences were found for TGFβ2 and ‐3 mRNA (p=1·0; p=0·2, respectively; Fishers exact test). In addition, mRNA analysis performed by RT‐PCR in seven cases (five high‐grade and two low‐grade osteosarcomas) confirmed the presence of high levels of TGFβ1 in high‐grade osteosarcomas, while low‐grade tumours had low or absent mRNA expression. In conclusion, this positive association suggests that TGFβ1 may be involved in determining the aggressive clinical behaviour of high‐grade osteosarcomas.

Epstein-Barr Virus (EBV) Infection and Undifferentiated Carcinoma of the Parotid Gland in Caucasian Patients

The Epstein-Barr virus (EBV) has been detected in certain types of lymphoma and some epithelial neoplasms such as nasopharyngeal lymphoepithelioma and occasional undifferentiated carcinomas in several organs including the salivary glands. However, clonal EBV genomes have been detected in undifferentiated carcinomas of the parotid gland exclusively in Alaskan natives and Eskimos, both groups being at the highest risk for nasopharyngeal carcinoma. The authors investigated the possibility that EBV may be present in undifferentiated parotid carcinomas in Caucasian subjects. To test this hypothesis, in situ hybridization (ISH) technique with biotinylated EBV-DNA probes was utilized on routinely processed, paraffin-embedded tissues from 7 cases of undifferentiated carcinomas of the parotid gland. EBV genomes were demonstrated in the cytoplasm of tumor cells from 3 out of 7 specimens tested. Surprisingly, EBV genomes were found in 3 out of 5 (60%) undifferentiated carcinomas that had developed in patients with a history of a long-persisting asymptomatic parotid mass, which had suddenly increased in size. Conversely, none of the undifferentiated carcinomas with continuous and rapid growth studied was found to be positive for EBV-DNA by ISH technique. Taken together, these data might suggest a possible role of EBV in the transformation of benign parotid gland lesions into malignant and aggressive undifferentiated carcinoma of the parotid gland, the so-called carcinoma expleomorphic adenoma.

Lack of prognostic value of p53 protein expression in node-negative breast cancer.

Aims and background The association of p53 protein accumulation and prognosis in node-negative breast cancer patients has been alternately demonstrated and denied in literature reports, and opinions on the use of p53 expression as an indicator of high risk of recurrence and as a guide for adjuvant therapy are controversial. Study design The association of p53 protein accumulation with prognosis was retrospectively evaluated in a series of 221 node-negative breast cancer patients treated with surgery alone and followed up for a minimum of 10 years. p53 accumulation was determined by immunohistochemistry on archive material, and classified into four grades of increasing immunostaining. Results No association was observed between p53 and age or pT category, whereas a significant association with nuclear grade was found (P = 0.0014). Univariate and multivariate analysis of 10-yr disease-free and overall survival showed a significant and independent prognostic association for tumor size (pT category) and nuclear grading but not for p53 expression, whatever grade grouping was used. Conclusions We did not find any evidence supporting the use of p53 immunostaining in current practice as an independent prognostic indicator or as a discriminant factor for adjuvant treatment of node-negative breast cancer patients.

Lack of Detection of Human Papillomavirus (HPV) in Transformed Laryngeal Keratoses by in situ Hybridization (ISH) Technique

Laryngeal keratosis (LK) is a precancerous mucosal change with a variable possibility of malignant transformation. Recent studies evidencing HPV-DNA genomes in a large series of non-malignant and malignant laryngeal lesions suggest a role of HPV in the transformation of laryngeal lesions possibly in synergistic interaction with other carcinogens. In this study, we analyzed 115 biopsy specimens from benign laryngeal lesions to evaluate the risk of malignant transformation and its relationship to degree of dysplasia and to histological features of virus cell infection. The rate of transformation of LK was 8% (9/115). Our results indicate that the risk of transformation in laryngeal keratoses without dysplasia (LKWOD) is lower than that in laryngeal keratoses with dysplasia (2.2% vs 25%, respectively) (p < 0.05). An increased risk of malignant evolution in laryngeal keratoses with dysplasia (LKWD) was also related to the degree of dysplasia (rate of transformation of 12.5, 22.2 and 36% in mild, moderate and severe dysplasia, respectively). Histological features suggesting HPV infection (koilocytic-like atypia and epithelial papillary hyperplasia) were found in 6 LK only, no case subsequently developing cancer. In both benign and transformed LK, analyzed by ISH, we failed to detect HPV genomes, suggesting a major role of others carcinogens, such as tobacco and/or alcohol, in the transformation of LK.

Th1, Th2 and Th3 cytokines in the pathogenesis of bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune bullous skin disease mediated by autoantibodies against hemidesmosomal proteins. In addition to humoral immunity, the contribute of infiltrating T-helper (Th) autoreactive lymphocytes and their related cytokines to the pathomechanism of blistering is now growing in interest. To investigate T-cell activation markers and the presence of inflammatory and fibrogenic cytokines (i.e. IL2, IL-4, IL-5, IFN-gamma, TGF-beta) in BP lesional skin, we performed an immunohistochemical study and an in situ hybridization procedure on five BP patients, comparing them with two psoriatic patients and four healthy subjects. Our aim was to expand suitable information about tissutal expression of cytokines, secondly to further investigate the role of TGF-beta (a Th3-like or T-regulatory (T-reg) cytokine) in a non-scarring disorder like BP, in order to highlight its pleiotropic activity. The immunohistochemical analysis revealed a moderate to strong staining for IL-4 and IL-5 with a prevalent perivascular localization in the upper dermis. The staining for IFN-gamma showed a moderate/focal expression on the dermal perivascular infiltrate. IL-2 protein was observed in four cases. While no positive staining for IL-4 mRNA was detected in all BP subjects with in situ hybridization, IL-5 mRNA was documented in four BP specimens. A focal nuclear staining for IFN-gamma was observed in the epidermal layers and on the cellular infiltrate of lesional skin. In all BP cases, a moderate/diffuse positivity for TGF-beta(1) mRNA was documented in both cytoplasm and nucleus of the infiltrating perivascular cells of lesional and perilesional skin. Our results suggest a balance between Th1, Th2 and Th3 activity, with quantitatively different impact of the various cytokines on the pathomechanism of blistering, depending on the reciprocal network. The supposed participation of each cytokine analyzed in the pathogenesis of BP is discussed. The newest data obtained consist of TGF-beta detection in a non-scarring disease like PB, that had never been documented before, and in the confirmation of a mixed cytokine pattern in the fully developed phase of the disease.

IL-4, IL-5, TGF-β1 and IFN-γ mRNAs detected by a new in situ amplification system in cicatricial pemphigoid

Abstract: The process that induces chronic progressive scarring in cicatricial pemphigoid (CP), a rare group of autoimmune mucocutaneous blistering diseases, is still under investigation. The tendency to heal with scar formation observed in CP could be due to the specific localization of the antigen in the basement membrane zone or could depend on the frequent recurrence of the disease in a localized area. The release of soluble fibrogenic factors by inflammatory infiltrating cells has also been considered as pathogenetically relevant. The aim of this study is to evaluate the expression of mRNA for IL‐4, IL‐5, TGF‐β1, IFN‐γ in patients with CP, and investigate the role of the cytokine profile as a possible cause of the clinical features and course of the disease. Fourteen patients (3 male, 11 female; age range 40–72 years) with oral (n = 10), preputial (n = 3) and cutaneous (n = 1) CP were studied. The formalin‐fixed and paraffin‐embedded biopsies were examined by in situ hybridization performing a new amplification system based on biotinyl‐tyramide. As a control, 4 patients (2 male, 2 female; age range 58–73 years) affected by bullous pemphigoid (BP), the most common autoimmune subepidermal blistering disease, were also examined. In CP, IL‐4 mRNA expression was present in 4 out of the 14 cases analysed. IL‐5 was detected in 12 CP biopsies. TGF‐β1 and IFN‐γ mRNAs were identified in 9 and 11 CP cases, respectively. In BP, an IL‐4 hybridization signal could not be observed in any of the cases. By contrast IL‐5, TGF‐β1 and IFN‐γ mRNA analyses were positive in all four BP cases. Our results suggest the presence of a T‐cell population with a mixed cytokine pattern in the cellular infiltrate of both blistering diseases, with a corresponding increase of Th2‐like activity in fully developed lesions, irrespective of the different sites involved. In addition, on the basis of the constant presence of TGF‐β1 mRNA in the different lesional phases of CP, and its overlapping expression in BP, we hypothesize that the involvement of additional factors is responsible for the scarring course typical of CP.

p53 protein overexpression in a case of conjunctival micro-invasive carcinoma.

We analysed by immunohistochemistry the expression of p53 protein in a case of micro-invasive carcinoma of the conjunctiva. About 50% of tumor cells showed a strong nuclear positivity for p53. This suggests that p53 gene alterations play a role in the development of this type of tumor.