Stefania Uccini

RAS signaling dysregulation in human embryonal Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation‐positive and mutation‐negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.

Serologic and genetic markers of celiac disease: A sequential study in the screening of first degree relatives

Objectives: The prevalence of celiac disease (CD) among the relatives and the complications of an undiagnosed CD prompted us to identify a useful disease screening strategy. Methods: We studied 441 first degree relatives of 208 CD patients by immunoglobulin (Ig)A antiendomysium antibodies (EMA) and radioimmunoprecipitation assay (RIA) IgA antitransglutaminase autoantibodies (TGAA). Of these, 364 were typed for human leukocyte antigen-DRB1, -DQA1, and -DQB1 genes by the polymerase chain reaction sequence specific primers method. It was suggested to the autoantibody-positive subjects that they should undergo intestinal biopsy. Results: TGAA were positive in 46 of 439 relatives, EMA in 38; intestinal lesions related to CD were present in 40 subjects. We also found two immunodeficient fathers with duodenal villous atrophy. In three serology-positive subjects, permission for intestinal biopsy was refused; for another three serology-positive cases, duodenal mucosa was normal. Thus, the strict CD prevalence resulted 9.5%, the enlarged prevalence 10.9%. The DQ2/DQ8 heterodimers were carried in 231 of 364 subjects and in 38 of 40 biopsy-proven celiac patients. Three DQ2-positive parents became positive to the serology during a long-lasting follow-up. Conclusions: On the basis of a carefully conducted study, CD prevalence in our series was seen as very high. These data suggest an accurate algorithm to select candidates for intestinal biopsy among CD high-risk subjects. First, an evaluation of the sensitive RIA TGAA and of total IgA (in IgA deficiency RIA IgG anti-tissue transglutaminase assay) should be performed. Then, an evaluation of the TGAA and the genetic study would be advisable 2 to 3 years later in negative subjects. Those carrying the DQ2/DQ8 heterodimers should continue the serologic follow-up; the others need a clinical follow-up.

Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization

Cellular distribution of HIV‐1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV‐1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell‐free virus is suggested. In the testes of AIDS‐deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV‐seropositive men produce infected spermatozoa that are released in the genital tract.—Muciaccia, B., Uccini, S., Filippini, A., Ziparo, E., Paraire, F., Baroni, C. D., Stefanini, M. Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization. FASEB J. 12, 151–163 (1998)

Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread

We studied the immunohistochemical expression of inducible adhesion molecules, integrins and extracellular matrix proteins in 10 cases of glioblastoma multiforme in order to investigate their angiogenesis, local invasiveness, poor metastasizing properties and their lack of tumour infiltrating leukocytes. In glioblastomas endothelial proliferations represent the majority of vascular structures; they were positive for endothelial markers (vWF, CD31, VE‐cadherin) and negative for macrophage markers (CD68, PAM‐1). Immunohistologically, they were subtyped into: 1 solid‐glomeruloid ICAM‐1, α2β1, α3β1, α5β1 negative; 2 channelled‐branching ICAM‐1 negative and α2β1, α3β1, α5β1 positive; 3 channelled‐telangiectatic ICAM‐1, α2β1, α3β1, α5β1 positive. In channelled proliferations, the expression and distribution of tenascin and merosin in the basal membrane was similar to that of normal brain vessels. The expression of all these molecules might indicate different steps of maturation of endothelial proliferations. The majority of endothelial proliferations may be immunohistologically considered as incomplete vascular structures; this might account for the low metastasizing tendency and low recruitment of leukocytes by these tumours. Neoplastic astrocytes were GFAP‐1, ICAM‐1, VCAM‐1, α2β1, α3β1 and α5β1 immunoreactive and α6β4 negative; this allows them to interact with extracellular matrix proteins and might, in part, explain the tendency of glioblastomas to infiltrate locally.

Circulating spindle cells : correlation with human herpesvirus-8 (HHV-8) infection and Kaposi's sarcoma

Vol 349 • January 25, 1997 255 cultures with the endothelial cell marker VE-cadherin and the tissue-macrophage marker PAM-1 (40–60% in KS patients and 0·5–6% in controls) (table). 4 weeks later, culture were examined for HHV-8 DNA sequences by nested PCR. None of the control cultures was positive, whereas HHV-8 was detected in all T-KS and AIDS-KS cases and in 11/14 cultures derived from patients with C-KS (table). HHV-8 was also detected in the patient with C-KS after bleomycin therapy. Our results indicate that an expansion of circulating KS-like cells or their progenitors is present in all forms of KS; KS-derived cultures are infected by HHV-8; and the presence of HHV-8 in these cells correlates with KS. Since these cells are capable of chemotaxis and can induce KS-like lesion formation in nude mice, our data suggest that they may localise into tissues, transmit HHV-8 infection to neighbour cells, and participate in the formation of KS lesions.

The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

ABSTRACT Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitts lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

Expression of ICAM-1 and VCAM-1 in human malignant mesothelioma

Intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) are cytokine‐inducible adhesion molecules which recognize ligands that are highly expressed on leukocytes. Expression of ICAM‐1 and VCAM‐1 was investigated in tissue sections of 16 cases of malignant mesothelioma (seven epithelial, eight biphasic, and one sarcomatoid) using immunohistochemistry. Neoplastic cells were diffusely and intensely stained for ICAM‐1 in all cases. VCAM‐1 was detected in 14 of 16 cases. The percentage of VCAM‐1‐positive tumour cells was more than 50 per cent in eight cases and the staining was observed mainly in epithelial‐like cells. VCAM‐1 was rarely expressed in other malignant tumours of epithelial origin, being present in only 1 of 58 cases of carcinoma originating from different anatomical sites. At the cellular level, ICAM‐1 and VCAM‐1 appeared co‐distributed, the staining for both being cytoplasmic with a membrane reinforcement. The regulation of VCAM‐1 expression by neoplastic mesothelial cells was investigated in vitro using 14 mesothelioma cell lines. ICAM‐1 was expressed by cultured cells of all mesothelioma cell lines, even in the absence of cytokines. VCAM‐1 was detected in 10–50 per cent of the cells in three non‐stimulated mesothelioma cell lines (mero‐95, mero‐96, and mero‐134), and was absent or poorly expressed in the remaining 11. Exposure of a negative cell line (mero‐48a) to an optimal concentration of tumour necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13) for 6–18 h resulted in the induction of VCAM‐1 mRNA synthesis and in VCAM‐1 expression at the membrane level in 60–70 per cent of the cells. These findings are consistent with the possibility that TNFα, IL‐13, or other activating signals are released in the tumour micro‐environment and regulate the expression of VCAM‐1 in malignant mesothelioma cells.

Clinical Significance of CXC Chemokine Receptor-4 and c-Met in Childhood Rhabdomyosarcoma

Purpose: The CXC chemokine receptor-4 (CXCR4)/stromal-derived factor-1 and c-Met/hepatocyte growth factor axes promote the metastatic potential of rhabdomyosarcoma cell lines in experimental models, but no data are available on their role in rhabdomyosarcoma tumors. The expressions of CXCR4 and c-Met were evaluated in primary tumors and isolated tumor cells in marrow, and were correlated with clinicopathologic variables and survival. Experimental Design: Forty patients with recently diagnosed rhabdomyosarcoma were retrospectively enrolled. CXCR4 and c-Met expression was investigated in primary tumors by immunohistochemistry, in isolated marrow-infiltrating tumor cells using double-label immunocytology. Results were expressed as the mean percentage of immunostained tumor cells. Results: CXCR4 and c-Met were expressed in ≥5% of tumor cells from 40 of 40 tumors, with 14 of 40 cases showing ≥50% of immunostained tumor cells (high expression). High CXCR4 expression correlated with alveolar histology (P = 0.006), unfavorable primary site (P = 0.009), advanced group (P < 0.001), marrow involvement (P = 0.007), and shorter overall survival and event-free survival (P < 0.001); high c-Met expression correlated with alveolar histology (P = 0.005), advanced group (P = 0.04), and marrow involvement (P = 0.02). In patients with a positive diagnosis for isolated tumor cells in marrow (n = 16), a significant enrichment in the percentage of CXCR4-positive (P = 0.001) and c-Met–positive (P = 0.003) tumor cells was shown in marrow aspirates compared with the corresponding primary tumors. Conclusions: CXCR4 and c-Met are widely expressed in both rhabdomyosarcoma subtypes and, at higher levels, in isolated marrow-infiltrating tumor cells. High levels of expression are associated with unfavorable clinical features, tumor marrow involvement and, only for CXCR4, poor outcome. In rhabdomyosarcoma, CXCR4 and c-Met represent novel exploitable targets for disease-directed therapy.

Intratumoral microvessel density and expression of ED-A/ED-B sequences of fibronectin in breast carcinoma

The aim of this study was to examine the correlation between intratumoral microvessel density (iMVD) and the presence of cellular fibronectin isoforms, ED-A and ED-B, in order to identify those tumours with a prominent angiogenic phenotype. 91 cases of invasive ductal breast carcinoma were evaluated for TNM, histological grading, percentage of Ki-67+ cells and receptor hormonal status. iMVD was determined as a single microvessel count in a 200 x microscope field from the region of the tumour that appeared to be most densely vascular. When the mean values of iMVD of the various groups were compared, no significant difference was noted (Mann-Whitney test). When tumours were classified as high or low iMVD, based on a cut-off value (99 vessels/0.74 mm2), cases with high iMVD were significantly more numerous in poorly differentiated G3 tumours (P = 0.01, Chi-square test), and in tumours with lymph node metastasis (N0 versus N1 + N2; P = 0.002). The possibility that high iMVD was the expression of prominent vascular neoformation was explored using ED-A and ED-B isoforms of fibronectin as markers of neoformed vessels. ED-A + and/or ED-B + blood vessels were < 10% of total vessels, were detected in approximately 50% of cases independently of iMVD values, and were not more numerous in tumour areas with hot spot vascularisation. Our findings indicate that iMVD and expression of ED-A/ED-B reflect different aspects of tumour-associated angiogenesis.

Immunoreactivity for S-100 protein in dendritic and in lymphocyte-like cells in human lymphoid tissues*

SummaryS-100 protein is an immunohistochemical marker for a subset of dendritic cells, the interdigitating reticulum cells (IDRCs), which are mainly located in T-dependent areas of lymphoid tissues. In the present study we have investigated the distribution of S-100-positive cells in lymph nodes, spleen, thymus and peripheral blood of normal subjects. Immunoreactivity for S-100 protein was demonstrated in large cells with dendritic morphology and in small lymphocyte-like cells present in the lymph node paracortex, thymic medulla, splenic periarterial lymphatic sheaths (PALS) and in peripheral blood. S-100-positive lymphocyte-like cells were frequently detected around high endothelial venules (HEV) and were present in numbers comparable to those of S-100-positive IDRCs. Immunoelectron microscopy confirmed the existence of positive cells with lymphoid morphology and revealed that the intracellular distribution of the immunoreaction product was similar in lymphoid and dendritic cells. Further characterization of S-100-positive cells demonstrated that both lymphoid and dendritic cells were unreactive with a large panel of monocytic and macrophage markers.