Gianna Baroni

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

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.

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.

PD-L1 marks a subset of melanomas with a shorter overall survival and distinct genetic and morphological characteristics

BACKGROUND Programmed cell death ligand 1 (PD-L1) is a cell surface molecule that plays a critical role in suppressing immune responses, mainly through binding of the PD-1 receptor on T lymphocytes. PD-L1 may be expressed by metastatic melanoma (MM). However, its clinical and biological significance remains unclear. Here, we investigated whether expression of PD-L1 in MM identifies a biologically more aggressive form of the disease, carrying prognostic relevance. PATIENTS AND METHODS PD-L1 expression was analyzed by immunohistochemistry using two different antibodies in primary tumors and paired metastases from 81 melanoma patients treated at a single institution. Protein expression levels were correlated with PD-L1 mRNA, BRAF mutational status and clinical outcome. PD-L1(+) and PD-L1(-) subsets of the A375 cell line were stabilized in vitro and compared using gene expression profiling and functional assays. Results were confirmed using xenograft models. RESULTS PD-L1 membrane positivity was detected in 30/81 (37%) of patients. By multivariate analysis, Breslow thickness and PD-L1 membrane positivity were independent risk factors for melanoma-specific death {PD-L1 5% cutoff [hazard ratio (HR) 3.92, confidence interval (CI) 95% 1.61-9.55 P < 0.003], PD-L1 as continuous variable (HR 1.03, 95% CI 1.02-1.04 P < 0.002)}. PD-L1 expression defined a subset of the BRAF-mutated A375 cell line characterized by a highly invasive phenotype and by enhanced ability to grow in xenograft models. CONCLUSIONS PD-L1 is an independent prognostic marker in melanoma. If confirmed, our clinical and experimental data suggest that PD-L1(+) melanomas should be considered a disease subset with distinct genetic and morpho-phenotypic features, leading to enhanced aggressiveness and invasiveness.

Utility of CDX-2 in distinguishing between primary and secondary (intestinal) mucinous ovarian carcinoma: an immunohistochemical comparison of 43 cases.

Primary and secondary mucinous tumors can involve the ovaries and have similar histologic appearances. The differential diagnosis is important for surgical and chemotherapeutic treatment and for the prognosis, but often it is extremely difficult. This article discusses an immunohistochemical panel that includes carcinoembryonic antigen (CEA), cytokeratin (CK) 7, CK20, CA125, CA19.9, and a new marker, CDX-2, for the distinction between primary ovarian mucinous carcinomas and metastatic (intestinal) ovarian tumors. Forty-three cases representing primary and secondary ovarian tumors were considered and consisted of 14 primary mucinous ovarian carcinomas (PMOCs) and 29 secondary (intestinal) ovarian tumors (SI-OTs). Fisher exact test was performed to evaluate the reliability of the respective antibodies to discriminate between PMOCs and SIOTs. CDX-2 was diffusely positive in all SIOTs and was expressed focally in 3 cases (21.42%) of PMOCs. CK7 was diffusely positive in 13 cases (44.82%) of SIOTs and in 13 cases (92.85%) of PMOCs. CK20 was diffusely positive in 17 cases (58.62%) of SIOTs and in 6 cases (42.85%) of PMOCs. CEA was diffusely positive in 28 cases (96.55%) of SIOTs and in 12 cases (85.71%) of PMOCs. CA19.9 was positive in all SIOTs and in 12 cases (85.71%) of PMOCs. CA125 was positive in 3 cases (10.34%) of SIOTs and in 4 cases (28.57%) of PMOCs. CK7 and especially CDX-2, a specific and sensitive marker, can aid pathologists in making a differential diagnosis (P = 0.003 and P < 0.0005, respectively), whereas CEA, CK20, CA125, and CA19.9 markers are not high enough to distinguish between primary and secondary mucinous ovarian tumors.

The status of PD-L1 and tumor-infiltrating immune cells predict resistance and poor prognosis in BRAFi-treated melanoma patients harboring mutant BRAFV600

BACKGROUND BRAF inhibitors (BRAFi) improve survival in metastatic melanoma patients (MMP) but the duration of clinical benefit is limited by development of drug resistance. Here, we investigated whether the expression of programmed death-ligand 1 (PD-L1) and the density of tumor-infiltrating mononuclear cells (TIMC) predict the occurrence of resistance, hence affecting the clinical outcome in BRAFi-treated MMP. METHODS PD-L1 expression (cutoff 5%) was analyzed by immunohistochemistry with two different antibodies in BRAF(V600)-mutated formalin-fixed and paraffin-embedded samples from 80 consecutive MMP treated with BRAFi at a single institution. TIMC were evaluated by conventional hematoxylin and eosin staining. RESULTS Forty-six and 34 patients received vemurafenib and dabrafenib, respectively. Membranous expression of PD-L1 was detected in 28/80 (35%) of patients. At multivariate analysis, absence of tumoral PD-L1 staining [odd ratio (OR) 10.8, 95% confidence interval (CI) 2.7-43.3, P < 0.001] and the presence of TIMC (OR 6.5, 95% CI 1.7-24.3, P < 0.005) were associated with a better response to treatment. Median progression-free survival (PFS) and overall survival were 10 and 15 months, respectively. By multivariate assessment, PD-L1 expression [hazard ratio (HR) 4.3, 95% CI 2.1-8.7, P < 0.0001] and absence of TIMC (HR 2.5, 95% CI 1.4-4.7, P < 0.002) correlated with shorter PFS. PD-L1 overexpression (HR 6.2, 95% CI 2.8-14.2, P < 0.0001) and absence of TIMC (HR 3.1, 95% CI 1.5-6.5, P < 0.002) were independent prognostic factors for melanoma-specific survival. CONCLUSION Our results provide the first proof-of-principle evidence for the predictive and prognostic relevance of PD-L1 immunohistochemical expression and density of immune cell infiltration in BRAF(V600)-mutated MMP treated with BRAFi.

β-adrenoceptors are upregulated in human melanoma and their activation releases pro-tumorigenic cytokines and metalloproteases in melanoma cell lines.

Recent studies sight β-adrenergic receptor (AR) antagonists as novel therapeutic agents for melanoma, as they may reduce disease progression. Here within, we evaluated the expression of β-ARs in a series of human cutaneous melanocytic lesions, and studied the effect of their endogenous agonists, norepinephrine (NE) and epinephrine (E), on primary and metastatic human melanoma cell lines. Using immunohistochemistry, we found that both β1- and β2-ARs are expressed in tissues from benign melanocytic naevi, atypical naevi and malignant melanomas and that expression was significantly higher in malignant tumours. Melanoma cell lines (human A375 primary melanoma cell line and human Hs29-4T metastatic melanoma cell lines) also expressed β1- and β2-ARs by measuring transcripts and proteins. NE or E increased metalloprotease-dependent motility, released interleukin-6 and 8 (IL-6, IL-8) and vascular endothelial growth factor (VEGF). These effects of catecholamines were inhibited by the unselective β-AR antagonist propranolol. The role of soluble factors elicited by catecholamines seemed pleiotropic as VEGF synergized with NE increased melanoma invasiveness through 3D barriers, while IL-6 participated in stromal fibroblast activation towards a myofibroblastic phenotype. Our results indicate that NE and E produce in vitro via β-ARs activation a number of biological responses that may exert a pro-tumorigenic effect in melanoma cell lines. The observation that β-ARs are upregulated in malignant melanoma tissues support the hypothesis that circulating catecholamines NE and E, by activating their receptors, favour melanoma progression in vivo.

Abnormalities of GATA-1 in Megakaryocytes from Patients with Idiopathic Myelofibrosis

The abnormal megakaryocytopoiesis associated with idiopathic myelofibrosis (IM) plays a role in its pathogenesis. Because mice with defective expression of transcription factor GATA-1 (GATA-1(low) mutants) eventually develop myelofibrosis, we investigated the occurrence of GATA-1 abnormalities in IM patients. CD 34(+) cells were purified from 12 IM patients and 8 controls; erythroblasts and megakaryocytes were then obtained from unilineage cultures of CD 34(+) cells. Purified CD 61(+), GPA(+), and CD 34(+) cells from IM patients contained levels of GATA-1, GATA-2, and FOG-1 mRNA, as well as of GATA-2 protein, that were similar to controls. In contrast, CD 61(+) cells from IM patients contained significantly reduced GATA-1 protein. Furthermore, 45% of megakaryocytes in biopsies from IM patients did not stain with anti-GATA-1 antibody, as compared to controls (2%), essential thrombocythemia (4%), or polycythemia vera (11%) patients. Abnormalities in immunoreactivity for FOG-1 were not found, and no mutations in GATA-1 coding sequences were found. The presence of GATA-1(neg) megakaryocytes in bone marrow biopsies was independent of the Val 617 Phe JAK 2 mutation, making it unlikely that a downstream functional relationship exists. We conclude that megakaryocytes from IM patients have reduced GATA-1 content, possibly contributing to disease pathogenesis as in the GATA-1(low) mice and also representing a novel IM-associated marker.

Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.

Correlation of Epidermal Growth Factor Receptor Expression with Tumor Microdensity Vessels and with Vascular Endothelial Growth Factor Expression in Ovarian Carcinoma

We analyzed in advanced ovarian serous G3 carcinoma the correlation between epidermal growth factor receptor (EGFR) overexpression and tumor angiogenesis and their relation with clinical outcome. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) were statistically correlated with disease-free interval and death from disease both in univariate and multivariate analyses while EGFR expression was not correlated with clinical outcome. MVD was significantly associated with progression of disease during chemotherapy while VEGF and EGFR expression were not correlated with responsiveness to chemotherapy (Fisher’s exact test). VEGF expression was correlated with MVD (Fisher’s exact test). EGFR showed a trend to correlation with MVD. Further studies focusing on the use of angiogenesis inhibitors in addition to EGFR inhibitors on ovarian carcinoma cells may produce therapeutic strategies in the selection of tailored therapies in ovarian cancer patients.