Maria Giulia Cangi

High syndecan-1 expression in breast carcinoma is related to an aggressive phenotype and to poorer prognosis

Syndecan‐1 is a transmembrane heparan sulphate proteoglycan that is involved in cell–cell adhesion, organization of cell–matrix adhesion, and regulation of growth factor signaling.

Salvage therapy with temozolomide in patients with aggressive or metastatic pituitary adenomas: experience in six cases

OBJECTIVE The prognosis of either pituitary carcinoma or aggressive pituitary adenoma resistant to standard therapies is poor. We assessed the efficacy of treatment with temozolomide, an oral second-generation alkylating agent, in a consecutive series of six patients with aggressive pituitary adenomas. DESIGN This was a 1-year prospective study of temozolomide therapy in six consecutive patients with pituitary carcinoma (one case) or atypical pituitary adenoma (five cases) resistant to standard therapies. There were three males and three females. Age at enrollment ranged between 52 and 64 years. Temozolomide was given orally at a dose of 150-200 mg/m(2) per day for 5 days every 4 weeks for a maximum of 12 cycles. METHODS Response assessment was based on measurable change in tumor size, as assessed on magnetic resonance imaging, and hormone levels. Response was defined as reduction of at least 50% of tumor size and hormone levels. RESULTS Four patients completed the 12 cycles of temozolomide treatment, as planned. Two patients stopped the drug after 3 and 6 months respectively because of the progression of disease. Two patients responded to temozolomide, while the remaining two patients had stable disease. Immunohistochemistry for O(6)-methylguanine-DNA methyltransferase (MGMT) in tumor sample showed a partial association with treatment response. CONCLUSIONS Temozolomide treatment has a wide range of efficacy in patients with pituitary carcinoma or locally aggressive pituitary adenoma. Positive staining for MGMT seems likely to predict a lower chance of response.

Chlamydia Infection and Lymphomas: Association Beyond Ocular Adnexal Lymphomas Highlighted by Multiple Detection Methods

Purpose:Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamydia detection tools to identify Cp elementary bodies–containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. Experimental Design: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. Results: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact Cp elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyers ring. Conclusions: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated.

Alterations of β-Catenin Pathway in Non-Melanoma Skin Tumors: Loss of α-ABC Nuclear Reactivity Correlates with the Presence of β-Catenin Gene Mutation

To determine the role of β-catenin pathway in human skin carcinogenesis, 135 non-melanoma skin tumors were analyzed for β-catenin expression and gene mutations. Intense nucleo-cytoplasmic immunoreactivity for C terminus β-catenin antibodies was observed in all pilomatricomas and in single cases of trichoepithelioma and squamous cell carcinoma showing peculiar signs of matrical differentiation. Moderate increase of β-catenin nuclear staining was detected in a significant proportion of basal cell carcinomas, Bowen disease, spiroadenomas, and occasionally also in squamous cell carcinomas, but in these neoplasms only a limited fraction of tumor cells accumulated β-catenin. Molecular analysis revealed that β-catenin gene mutations are a peculiar feature of skin tumors with matrical differentiation and correlate with a pattern of intense and diffuse β-catenin nuclear expression. In contrast, adenomatous polyposis coli (APC) and AXIN2 mutations were not involved in skin tumorigenesis. Analysis of Wnt pathway revealed that TCF-1 and MITF-M were selectively induced in the tumor types harboring β-catenin mutations, indicating that a Wnt/β-catenin pathway involving TCF-1 and MITF-M is activated in these tumors. Interestingly, high expression levels of TCF-3 were found in basal cell carcinomas and spiroadenomas. TCF-3 is reported to act as a negative modulator of β-catenin degradation pathway. Thus, the moderate increase of β-catenin nuclear staining detected in these tumor types might, at least in part, be due to a TCF-3-dependent mechanism. Finally, we found that the presence of β-catenin mutations significantly correlated with loss of nuclear immunoreactivity for an antibody raised against the N terminus of β-catenin (αABC). Thus, a combined analysis with C terminus-β-catenin antibodies and αABC Ab may represent a powerful investigative approach for the detection of β-catenin structural alterations.

BRAFV600E-mutation is invariably present and associated to oncogene-induced senescence in Erdheim-Chester disease

Objectives Erdheim-Chester disease (ECD) is a rare form of histiocytosis characterised by uncontrolled chronic inflammation. The oncogenic BRAFV600E mutation has been reported in biopsies in 19 out of 37 patients with ECD from the largest published cohort, but never found in the patients’ peripheral blood. Also, the role of the mutation in the pathogenesis of the disease has not been elucidated yet. BRAFV600E has been associated with oncogene-induced senescence (OIS), a protective mechanism against oncogenic events, characterised by the induction of proinflammatory pathways. Methods We verified the BRAF status in biopsies and peripheral blood from 18 patients with ECD from our cohort and matched controls by means of immunohistochemistry and of an ultrasensitive assay, based on the combination of a locked nucleic acid PCR and pyrosequencing. Droplet digital PCR was used to confirm the findings. We also evaluated the presence of senescence markers in ECD histiocytes. Results BRAFV600E mutation was present in all the biopsy and peripheral blood samples from patients with ECD and in none of the controls. ECD histiocytes and a fraction of circulating monocytes from patients with ECD showed signs of a constitutive activation of the MAPK pathway. Moreover, BRAF-mutated histiocytes expressed markers of OIS. Conclusions The oncogenic BRAFV600E mutation is present in biopsies and in the peripheral blood from all patients with ECD who were evaluated and is associated with OIS. These findings have significant implications for the pathogenesis, diagnosis and treatment of ECD.

Mucin expression pattern in pancreatic diseases: findings from EUS-guided fine-needle aspiration biopsies.

OBJECTIVES:Alterations in mucin (MUC) glycosylation and expression have been described in cancer. Endoscopic ultrasound-guided fine-needle aspiration (EUS–FNA) can provide material for molecular biology analysis. This study assessed the feasibility of evaluating MUC expression from material obtained by EUS–FNA and studied the profile of MUC expression in benign and malignant pancreatic lesions.METHODS:A total of 90 patients with solid or cystic pancreatic lesions underwent FNA. The aspirated material was used for cytological analysis and RNA extraction to assess the expression pattern of MUCs by reverse transcription-PCR with primers specific for the MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes.RESULTS:RNA extraction was successful in 81% of the biopsies. The prevalences of MUC1, MUC2, MUC4, and MUC7 in ductal adenocarcinoma were 57.7, 51.4, 18.9, and 73.0%, respectively. Fifty percent of benign lesions and neuroendocrine tumors (NETs), and 63% of intraductal papillary mucinous neoplasms (IPMNs) were positive for MUC1. Twenty-five percent of benign lesions, 86% of NETs, and 47% of IPMNs were positive for MUC2. Of NETs, 50% were positive for MUC1, and 14% were positive for MUC7. None of the benign lesions or NETs expressed MUC4. MUC7 expression was highly significant for adenocarcinoma (P=0.007) and borderline for IPMN (P=0.05). MUC7 was expressed in 37.5% of chronic pancreatitis cases.CONCLUSIONS:RNA can be extracted from samples obtained under EUS–FNA. MUC7 could serve as a potential biological marker to identify malignant lesions, especially pancreatic adenocarcinoma.

Chlamydophila psittaci is viable and infectious in the conjunctiva and peripheral blood of patients with ocular adnexal lymphoma: Results of a single‐center prospective case–control study

Ocular adnexal MALT lymphoma (OAML) is linked to Chlamydophila psittaci (Cp) infection. Viability and infectivity of Cp, demonstrated by growth in culture, has not been yet investigated in these patients. We conducted a single‐center prospective case–control study to assess the prevalence, viability and infectivity of Cp in 20 OAML patients and 42 blood donors registered in a 6‐month period. The presence of Cp in conjunctival swabs and peripheral blood mononuclear cells (PBMC) of patients and donors was assessed by TETR‐PCR and in vitro cultures. From an epidemiological point of view, OAML patients often resided in rural areas, and reported a history of chronic conjunctivitis and prolonged contact with household animals (85% vs. 38% of donors; p = 0.00001). Cp was detected in lymphoma tissue in 15 (75%) patients. Cp DNA was detected in conjunctival swabs and/or PBMC from 10 (50%) patients and in PBMC from 1 (2%) donor (p = 0.01). Viability and infectivity of Cp, demonstrated by growth in culture, were confirmed in conjunctival swabs and/or PBMC from 5 (25%) patients, but not in donors (p = 0.002). This prospective study demonstrates, for the first time, that Cp present in the conjunctiva and PBMC of OAML patients is capable to grow and be isolated in cell cultures. Cp infection is common in OAML patients and exceptional in blood donors. Epidemiological data of OAML patients (prolonged contact with household animals and chronic conjunctivitis) are consistent with Cp exposure risk.

Tumor-associated macrophages as major source of APRIL in gastric MALT lymphoma

Lymphoid hyperplasia of gastric mucosa associated with Helicobacter pylori (HP) infection represents a preneoplastic condition of the mucosa associated lymphoid tissue (MALT), which may evolve to a B-cell lymphoma. While it is well established that the initial neoplastic proliferation of B cells is antigen-driven and dependent on the helper activity of HP-specific T cells, it needs to be elucidated which cytokine or soluble factor(s) promote B-cell activation and lymphomagenesis. Herein, we originally report that gastric MALT lymphoma express high levels of a proliferation inducing ligand (APRIL), a novel cytokine crucial in sustaining B-cell proliferation. By immunohistochemistry, we demonstrate that APRIL is produced almost exclusively by gastric lymphoma-infiltrating macrophages located in close proximity to neoplastic B cells. We also show that macrophages produce APRIL on direct stimulation with both HP and HP-specific T cells. Collectively, our results represent the first evidence for an involvement of APRIL in gastric MALT lymphoma development in HP-infected patients.

Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation

Evidence from certain geographical areas links lymphomas of the ocular adnexa marginal zone B-cell lymphomas (OAMZL) with Chlamydophila psittaci (Cp) infection, suggesting that lymphoma development is dependent upon chronic stimulation by persistent infections. Notwithstanding that, the actual immunopathogenetical mechanisms have not yet been elucidated. As in other B-cell lymphomas, insight into this issue, especially with regard to potential selecting ligands, could be provided by analysis of the immunoglobulin (IG) receptors of the malignant clones. To this end, we studied the molecular features of IGs in 44 patients with OAMZL (40% Cp-positive), identifying features suggestive of a pathogenic mechanism of autoreactivity. Herein, we show that lymphoma cells express a distinctive IG repertoire, with electropositive antigen (Ag)-binding sites, reminiscent of autoantibodies (auto-Abs) recognizing DNA. Additionally, five (11%) cases of OAMZL expressed IGs homologous with autoreactive Abs or IGs of patients with chronic lymphocytic leukemia, a disease known for the expression of autoreactive IGs by neoplastic cells. In contrast, no similarity with known anti-Chlamydophila Abs was found. Taken together, these results strongly indicate that OAMZL may originate from B cells selected for their capability to bind Ags and, in particular, auto-Ags. In OAMZL associated with Cp infection, the pathogen likely acts indirectly on the malignant B cells, promoting the development of an inflammatory milieu, where auto-Ags could be exposed and presented, driving proliferation and expansion of self-reactive B cells.

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon α-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.