Alessandro Franchi

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck

Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene.

Osteonectin expression correlates with clinical outcome in thin cutaneous malignant melanomas

Osteonectin, also termed BM40 or SPARC (secreted protein, acidic and rich in cysteine) is a multifunctional glycoprotein involved in tissue mineralization, cell-extracellular matrix interactions as well as angiogenesis. It has been suggested that osteonectin may play a key role in the process of tumoral invasion and metastasis in certain malignancies. In this study, we reviewed the clinical records and the histopathologic slides of 188 thin cutaneous malignant melanomas (< or = 0.75 mm). Among them, 12 cases underwent progression and were selected for the study. Osteonectin expression was investigated by immunohistochemistry in these 12 patients and 24 matched controls who did not undergo progression. Osteonectin staining was correlated with clinical outcome and other clinicopathologic parameters. Progression-free and disease-specific survival rates were calculated with the Kaplan-Meier method and their differences were evaluated by the log rank test. Overall, immunoreactivity for osteonectin was found in 23 (63.8%) cases. Eighteen cases (50%) displayed staining in 1% to 50% of neoplastic cells whereas five cases (13.8%) showed a diffuse positivity in more than 50% of the tumor cells. Osteonectin expression was significantly correlated with risk of progression (P = .01), incidence of distant metastases (P = .005) and survival (P = .03). There was a higher incidence of osteonectin-positive tumors in cases that did experience regional lymph node metastases versus those cases that did not, but that difference did not reach statistical significance (P = .06). No significant correlation was found between osteonectin expression and other clinicopathologic features, including age, sex, site, histotype, Clarks level, presence of regression, presence of inflammatory response, and tumor growth phase. Our data showed that osteonectin expression is a predictor of clinical outcome in thin cutaneous melanomas.

Tumour lymphangiogenesis is a possible predictor of sentinel lymph node status in cutaneous melanoma: a case–control study

Background: Cutaneous melanoma spreads preferentially through the lymphatic route and sentinel lymph node (SLN) status is regarded as the most important predictor of survival. Aims: To evaluate whether tumour lymphangiogenesis and the expression of vascular endothelial growth factor C (VEGF-C) is related to the risk of SLN metastasis and to clinical outcome in a case–control series of patients with melanoma. Methods: Forty five invasive melanoma specimens (15 cases and 30 matched controls) were investigated by immunostaining for the lymphatic endothelial marker D2-40 and for VEGF-C. Lymphangiogenesis was measured using computer assisted morphometric analysis. Results: Peritumorous lymphatic vessels were more numerous, had larger average size, and greater relative area than intratumorous lymphatics. The number and area of peritumorous and intratumorous lymphatics was significantly higher in melanomas associated with SLN metastasis than in non-metastatic melanomas. No significant difference in VEGF-C expression by neoplastic cells was shown between metastatic and non-metastatic melanomas. Using logistic regression analysis, intratumorous lymphatic vessel (LV) area was the most significant predictor of SLN metastasis (p = 0.04). Using multivariate analysis, peritumorous LV density was an independent variable affecting overall survival, whereas the intratumorous LV area approached significance (p = 0.07). Conclusions: This study provides evidence that the presence of high peritumorous and intratumorous lymphatic microvessel density is associated with SLN metastasis and shorter survival. The intratumorous lymphatic vessel area is the most significant factor predicting SLN metastasis. The tumour associated lymphatic network constitutes a potential criterion in the selection of high risk patients for complementary treatment and a new target for antimelanoma therapeutic strategies.

Adhesion, growth, and matrix production by osteoblasts on collagen substrata

SummaryA number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

An Osteoclast-Rich Tumor of the Gastrointestinal Tract with Features Resembling Clear Cell Sarcoma of Soft Parts: Reports of 6 Casesof aGIST Simulator

Six cases are reported of an osteoclast-rich tumor of the gastrointestinal tract that should be segregated from GIST. Five of the cases were located in the small bowel and one in the stomach. The age of the patients ranged from 13 to 37 years. The tumors behaved aggressively, with metastases to regional lymph nodes, liver, and other intraabdominal sites. Microscopically, the tumor cells were medium-sized, predominantly oval, relatively monomorphic, diffusely immunoreactive for S-100-protein, and negative for CD1 17, CD34, HMB-45, and Mart-i. They were admixed with scattered osteoclast-like, multinucleated giant cells which were S-100-protein negative and KPIpositive. One case studied cytogenetically had the karyotype 46XX t(I2;22)(qI3;q12). The cases here reported are interpreted as examples ofa distinctive type of gastrointestinal neoplasm which shares somefeatures with clear cell sarcoma of soft parts (melanoma of soft parts), including in one case the chromosomal translocation that is characteristically associated with that entity.

Prognostic factors and outcomes for osteosarcoma: an international collaboration

We aimed to evaluate the prognostic significance of traditional clinical predictors in osteosarcoma through an international collaboration of 10 teams of investigators (2680 patients) who participated. In multivariate models the mortality risk increased with older age, presence of metastatic disease at diagnosis, development of local recurrence when the patient was first seen, use of amputation instead of limb salvage/wide resection, employment of unusual treatments, use of chemotherapeutic regimens other than anthracycline and platinum and use of methotrexate. It was also influenced by the site of the tumour. The risk of metastasis increased when metastatic disease was present at the time the patient was first seen and also increased with use of amputation or unusual treatment combinations or chemotherapy regimens not including anthracycline and platinum. Local recurrence risk was higher in older patients, in those who had local recurrence when first seen and when no anthracycline and platinum were used in chemotherapy. Results were similar when limited to patients seen after 1990 and treated with surgery plus combination chemotherapy. This large-scale international collaboration identifies strong predictors of major clinical outcomes in osteosarcoma.

Evidence for differential expression of Notch receptors and their ligands in melanocytic nevi and cutaneous malignant melanoma

The Notch signaling has been implicated in the regulation of self-renewal of adult stem cells and differentiation of precursors along a specific cell lineage, in normal embryonic development and organogenesis. There is also evidence that signaling through Notch receptors regulate cell proliferation and cell survival in several types of cancer, with opposing results depending on tissue context. No data are available in the literature concerning modulation of the expression of Notch receptors, and their ligands, in human cutaneous malignant melanoma. Here, we have investigated, for the first time, the expression of Notch-1, Notch-2, Jagged-1, Jagged-2 and Delta-like 1 proteins, by immunohistochemistry, in a series of benign and malignant human melanocytic lesions: five common melanocytic nevi, five ‘dysplastic nevi’ and 20 melanomas (five in situ, five T1–T2, five T3–T4 and five metastatic melanomas). We found that the expression of Notch-1 and Notch-2, as well as Notch ligands, was upregulated in ‘dysplastic nevi’ and melanomas as compared with common melanocytic nevi. These results indicate that the activation of Notch may represent an early event in melanocytic tumor growth and upregulation of Notch signaling may sustain tumor progression.

Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions.

Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.

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.

Sinonasal undifferentiated carcinoma, nasopharyngeal-type undifferentiated carcinoma, and keratinizing and nonkeratinizing squamous cell carcinoma express different cytokeratin patterns.

Sinonasal undifferentiated carcinoma (SNUC) is a highly aggressive malignant neoplasm that is often difficult to distinguish from other poorly differentiated carcinomas arising in the sinonasal tract. To search for a differential cytokeratin (CK) expression that could be useful for diagnostic purposes, we compared the expression of a large panel of CKs in a series of 6 SNUCs, 10 poorly differentiated squamous cell carcinomas (SCCs), 10 nonkeratinizing squamous cell carcinomas (NKSCCs), and 5 nasopharyngeal-type undifferentiated carcinomas (NPTCs). SCC, NKSCC, and NPTC frequently showed immunoreactivity for CK5/CK6, CK8, CK13, and CK19. In addition, SCC and NKSCC expressed CK14, which was not detected in NPTC, and SCC expressed CK7 (60% of cases) and CK4 (30% of cases), which were absent in NKSCC and NPTC. Three NKSCCs were associated with a Schneiderian papilloma, and the results of the immunostaining were similar in the two components, with the exception of CK4 and CK7, which were expressed by the papilloma and not by the carcinoma. In contrast to other carcinomas, SNUC was characterized by the exclusive expression of CKs of simple epithelia, such as CK8 (100% of cases), CK7 (50% of cases), and CK19 (50% of cases). Thus, there are significant differences in the pattern of CK expression between SNUC, SCC, NKSCC, and NPTC, which could be of diagnostic aid. Moreover, these findings support the hypothesis that SNUC is a separate entity from SCC and NPTC of the sinonasal tract.