Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model.

BACKGROUND: Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth. METHODS: Murine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis. RESULTS: We found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis. CONCLUSIONS: Our results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human.

Human papillomavirus DNA and p16 gene in squamous cell lung carcinoma.

AIM: To investigate the presence of human papillomavirus (HPV) DNA in squamous cell carcinoma (SCC) of the lung, and to examine the protein expression and genomic status of p16 and their correlation. MATERIALS AND METHODS: Fifty cases of surgically removed primary lung SCC were analyzed. HPV detection was performed by Polymerase Chain Reaction (PCR) of L1 region and E6/E7 region of high-risk viral genotype. p16 protein and gene analysis were carried out by immunohistochemistry and Fluorescence In Situ Hybridization (FISH), respectively. RESULTS: HPV DNA was found in two out of 50 cases (4%, p>0.05). In five cases, p16 protein expression was positive. The data showed that in 45/50 cases (90%, p<0.05) HPV DNA and p16 were both negative, in 2/50 cases (4%) both were positive, and in 3/50 (6%) cases, HPV DNA was negative and p16 positive. FISH analysis for p16 gene showed aneusomia of chromosome 9 with or without loss of p16 gene in all cases (100%, p<0.05). CONCLUSION: Our study shows that in pulmonary SCC, there is no association between the presence of HPV DNA and the expression of p16 protein. Furthermore, the loss of the p16 gene and the instability of chromosome 9 were frequently found in HPV DNA-negative cases.

Diagnostic implications of L1, p16, and Ki-67 proteins and HPV DNA in low-grade cervical intraepithelial neoplasia.

The expressions of p16, Ki-67, and L1 proteins and human papillomavirus DNA were investigated using polymerase chain reaction (HPV/PCR) and catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC/ISH) as potential molecular markers for the diagnosis and transforming potential of low cervical intraepithelial neoplasia (CIN1). Ki-67 and p16 protein expression increased linearly from control cases to more dysplastic cases (CIN1, CIN2, and CIN3), peaking in squamous cell carcinoma cases (P<0.05). In contrast, L1 expression was inversely correlated with malignant transformation. Patients with CIN1 were divided into 4 groups: L1p16, L1p16, L1p16, and L1p16, and the immunohistochemical results were combined with HPV/PCR, L1/PCR, and high-risk E6/E7 genome and CSAC/ISH data. Malignant transformation correlated with L1p16 patients (100% of CIN2, CIN3, and squamous cell carcinoma cases) and was evident in approximately 23% of CIN1 cases. In addition, the presence of HPV/DNA was evident in 52% of CIN1 cases, and within the L1p16 group. In 4 of 7 cases, the high-risk E6/E7 HPV genome was present and in 1 case it was integrated into the host DNA, as confirmed using CSAC/ISH. In patients with CIN1, investigating the presence of HPV/DNA using PCR and the presence of the high-risk E6/E7 genome is necessary to distinguish high-risk oncogenic patient groups from low-risk groups. This study highlights the importance of combining immunohistochemical analysis with HPV/PCR and CSAC/ISH to identify patients with CIN1 with a risk of neoplastic progression.

Frequency and role of HPV in the progression of epithelial dysplasia to oral cancer.

BACKGROUND: Human papillomavirus DNA (HPV DNA) and p16 and p53 protein expressions were investigated for their role in transforming dysplasia into squamous cell carcinoma of the oral cavity in a non-smoker and non-drinker patient group. MATERIALS AND METHODS: A total of 56 oral biopsies from non-smoker and non-drinker patients were analyzed. The specimens were grouped into three categories: group 1 included 31 cases of hyperplastic mucosa and mild dysplasia, group 2 included 14 cases of moderate and severe dysplasia, while group 3 comprised 11 cases of invasive squamous cell carcinomas. In all cases, immunohistochemical methods were performed to detect p16 and p53 protein expressions. The nested polymerase chain reaction for HPV (nested HPV-PCR) and the catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC-ISH) methods were applied for HPV DNA detection and typing of high-risk genotype. RESULTS: P16 protein, absent from all specimens of group 1, was especially noted in group 2 (92.86%) and in group3 (54.55%). Five out of 14 of group 2 cases (35.71%) and 3/11 (27.27%) of group 3 were HPV DNA positive. The HPVs detected were of both high-risk and low-risk genotype. The analysis of the relationship between HPV and p16 protein expression revealed that all the group 2 and 3 samples with HPV DNA, overexpressed p16 protein. CONCLUSION: The results suggest that HPV could be a molecular marker in group 2 and 3 specimens in non-smoker and non-drinker patients. The virus may play an etiological role in carcinogenesis in the oral cavity. The association between HPV and p16 overexpression suggests a molecular mechanism similar to that found in cervical cancer.

HER family receptors expression in squamous cell carcinoma of the tongue: study of the possible prognostic and biological significance.

OBJECTIVES: The squamous cell carcinoma of the tongue (SCCT) is biologically and epidemiologically distinct from other oral cavity cancers and is associated with lower overall survival rates. The role of HER family members (HER-1, HER-2/neu, HER-3 and HER-4) in the pathogenesis and progression of head and neck squamous cell carcinomas has been demonstrated but no report have focused on SCCT. This study investigated, the expression of all members of the HER family, in a series of SCCT and studied the possible prognostic value and correlation with various clinico-pathological parameters. METHODS: HER-1, HER-2/neu, HER-3 and HER-4 expression was analysed by semi-quantitative immunohistochemical staining on paraffin embedded tissue specimens from 40 patients who underwent surgery for SCCT between 1996 and 2006. RESULTS: HER-1 was overexpressed in 26 cases (65%), HER-2/neu in two (5%), HER-3 in 19 (48%) and HER-4 in three cases (8%). No significant correlation was found between clinicopathological variables and expression of HER-1 and HER-2/neu. HER-3 overexpression was significantly related to nodal stage, age (>or=64 years) and decreased overall survival (P

Evaluation of the cytotoxic and genotoxic effects of orthodontic bonding adhesives upon human gingival papillae through immunohistochemical expression of p53, p63 and p16.

BACKGROUND: Numerous in vitro studies have shown that composite materials, commonly used for restorations in conservative dentistry, and in orthodontics to anchor brackets to the tooth enamel, have cytotoxic and genotoxic effects. The study determined expression of p53, p63 and p16, biomarkers useful for predicting potential genotoxicity. PATIENTS AND METHODS: p53, p63 and p16 expression was determined immunohistochemically in the gingival papillae of 99 patients (69 banded orthodontically for at least one year, brackets bonded to teeth with filled flowable composite resin, 30 without orthodontic banding as controls). The papillae samples were removed surgically and examined to evaluate morphological and biological alterations. RESULTS: In no case were morphological alterations visible by microscopy out of the 69 banded patients; four (5.80%) were positive for p53 and two for p63 expression in the basal and suprabasal layers (2.90%). One patient was positive for p16 (1.45%). No control case was positive for any of the biomarkers (0.00%). CONCLUSION: The significance of p53, p63 and p16 positivity, and whether these proteins may serve as biomarkers to predict the risk of developing oral lesions (dysplasia, oral cancer) is still unclear. Although details of the mechanisms leading to cell death, genotoxicity and cell-cycle delay are not fully understood, resin monomers may alter cell function in the oral cavity.

Expression of p16, p53 and Ki-67 proteins in the progression of epithelial dysplasia of the oral cavity.

BACKGROUND: The overexpression of the protein products of genes associated with the cell cycle tumour protein53 (p53), cyclin-dependent kinase inhibitor 2A (p16) and antigen identified by monoclonal antibody Ki-67 (Ki-67) is apparently of great significance. This study evaluated the immunohistochemical expression of these proteins in precancerous lesions and in carcinoma of the oral cavity. MATERIALS AND METHODS: The nuclear expression of p53 and Ki-67 and nuclear and/or cytoplasmic expression of p16 protein was examined in 54 biopsy specimens from the oral cavity obtained over a period of 3 years. The samples included 18 cases of normal/hyperplastic mucosa, 25 cases of dysplasia and 11 cases of invasive squamous cell carcinoma. The specimens were grouped into three categories: 1 = no or mild dysplasia, 2 = moderate or severe dysplasia, and 3 = invasive carcinoma. RESULTS: p16 was negative in all the group 1 specimens, while both p53 and Ki-67, when present, were limited to the cells of the basal layer. In the group 2 specimens, the number of p16-, p53-, and Ki-67-positive cells increased as the grade of dysplasia progressed. In group 3 (invasive carcinomas), p53 and p16 expression occurred respectively in 81.8% and 54.5% of cases, while Ki-67 was elevated in all the cases. CONCLUSION: The expression of the cell-cycle proteins p16 and p53 in the dysplastic epithelium, in association with Ki-67, may represent significant markers to recognize evolution of precancerous disease in the oral cavity and to improve identification of the degree of dysplasia.

Transforming growth factor-beta1 and CD105 promote the migration of hepatocellular carcinoma-derived endothelium.

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-beta1 (TGF-beta1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-beta1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-beta1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-beta1, Ve-cadherin (Ve-cad), CD44, beta-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-beta1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of TGF-beta1 was blocked by anti-CD105 antibodies and correlated with the grade of HCC malignancy. Histologic examination of HCC biopsies showed that HCCs with the worse malignant features had the highest expression of TGF-beta1, CD105, and angiogenic markers (Ve-cad and CD44). Because CD105 was highly expressed in microvessels at the tumor periphery and TGF-beta1 staining was only found in neoplastic hepatocytes, we conclude that HCC-derived TGF-beta1 may act as a chemoattractant for CD105-expressing ECs and as a promoter of tumor angiogenesis. Thus, drugs that selectively target the TGF-beta1/CD105 axis may interfere with HCC-related angiogenesis and HCC progression.

Comparative analysis of c-erbB-2 (HER-2/neu) in squamous cell carcinoma of the tongue: does over-expression exist? And what is its correlation with traditional diagnostic parameters?

OBJECTIVES: Over-expression of the proto-oncogene c-erbB-2 (HER-2/neu) has been shown to be a prognostic marker in many kinds of cancer, whereas conflicting data exist about the prevalence of HER-2/neu over-expression in squamous cell carcinoma (SCC) of the tongue. The status of Her-2/neu was evaluated in a series of SCC of the tongue to verify the frequency of over-expression of HER-2/neu and evaluate the correlation with traditional diagnostic parameters of this neoplasm. METHOD: Fourty patients with SCC of the tongue were investigated for over-expression of the protein through immunohistochemistry using CB11 antibody, the Hercep Test kit and FISH. RESULTS: Data obtained using the Hercep Test differ from published reports concerning the over-expression of Her-2/neu and there was no correlation between levels of expression of Her-2/neu and other clinico-pathological and/or prognostic parameters. Of the 40 specimens, using CB11 we obtained results in line with published reports; however, with the Hercep Test we found only 1 positive case (2.5%) (score 3+). CONCLUSION: These data, confirmed by FISH, suggest that Her-2/neu is not a suitable marker that could play a primary role in the clinical-therapeutic management of SCC of the tongue.

Different expression of HER2/neu oncogene in breast carcinoma and in liver metastasis. Description of a case.

We report the clinical, morphological and molecular findings regarding a 37-year-old woman with breast cancer metastatic to the liver and describe the different expression of a tumor marker in the primary and secondary lesions and the singular responsiveness to treatment. The patient suffered from a carcinoma of the left breast with metastasis to the liver. High HER-2 protein expression assessed by immunohistochemistry and HER-2/neu amplification determined by FISH were present in the primary tumor, while the liver metastasis showed a lower value of HER-2 protein (2+) and absence of HER-2/neu amplification. The patient was treated with chemotherapy (epirubicin and paclitaxel) followed by trastuzumab and docetaxel. After 5 months, at the completion of chemo-immunotherapy, liver ultrasonography showed a further hepatic response. A second biopsy was performed on the residual liver nodule: immunohistochemistry revealed negative (1+) HER-2 expression and FISH confirmed that the HER-2/neu gene was not amplified. The different amplification of HER-2 and expression of its protein in primary and metastatic carcinoma could be important for planning adequate treatment.