Vittoria Donofrio

MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

Background Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Conclusions/Significance Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

Prognostic factors in pleuro‐pulmonary blastoma

To evaluate the prognostic factors in a series of children affected by pleuropulmonary blastoma (PPB).

p53 protein in aggressive and non-aggressive basal cell carcinoma

Basal cell carcinoma (BCC) is the most frequent cutaneous neoplasm, with a generally favorable clinical behavior. Sometimes, indeed, it recurs after therapy and/or metastasizes. As point mutations in the coding sequence of the p53 tumor suppressor gene have been implicated in the progression of many human tumors, we studied the expression of p53 protein on this neoplasia. We tested immunohistochemically the positivity for p53 protein (NCL‐p53‐CM1, YLEM) on 19 cases of morphologically “non aggressive” BCC (BCC1) and on 19 “aggressive” BCC (BCC2), all with one or more relapses and 3 with distant meta‐stases also. Results were related to clinicopathological and follow‐up data. All but one BCC2 were found positive for p53 protein. Conversely, only 2 cases of BCC1 exhibited low immunoreactivity for p53 protein, with high statistical differences between the two groups. No correlation was found between the immunoreactivity, age of patients, and site of the lesions. The availability of immunohistochemistry and the relatively easy interpretation of the results make screening for p53 protein a possibly useful tool in the prognostic evaluation of BCC.

Ovarian Metastasis from Renal Cell Carcinoma: A Report of Three Cases

Ovarian metastases from renal cell carcinoma (RCC) are very rare. Over the past 16 years we have encountered 3 examples. The first 2 cases occurred in adults 49 and 50 years old, respectively, who had huge ovarian metastases, clinically detected 12 and 14 months, respectively, after diagnosis of RCC. The third case was a 17-year-old girl in whom a metastatic renal cell carcinoma was detected in an otherwise benign-appearing cystic ovarian mass. To the best of our knowledge only 11 cases of clinically detected ovarian metastases of RCC have been reported. We report 3 new cases and review the literature on the subject.

Sex cord-stromal tumors of the testis in children. A clinicopathologic report from the Italian TREP project

PURPOSE Testicular sex cord-stromal tumors (SCSTs) are very rare in children and include a variety of neoplasms with different clinical features and biologic behavior. Aim of the study was to report the clinical findings and results observed in a series of patients with testicular SCST, registered in a multi-institutional Italian network on rare tumors in children and adolescents. MATERIALS AND METHODS The records of 11 patients, enrolled in 6 Italian centers from January 2000 to December 2008, were reviewed. The Childrens Oncology Group (COG) staging system was adopted. Chemotherapy was recommended in patients with incomplete surgery or metastatic disease. RESULTS A testicular mass was the most common symptom. All patients underwent primary removal of the tumor; orchiectomy with high ligation of spermatic cord was performed in 7 and tumor enucleation in 4. At histology, 4 patients had Leydig cell tumors, 4 juvenile granulosa cell tumors, 1 Sertoli cell tumor, 1 incompletely differentiated SCST, and 1 SCST with an intermediate pattern Sertoli cell tumor/mixed form. The histology of 8 of 11 cases was reviewed and investigated through immunohistochemical stains. Ten children were in stage I; 1 patient, who did not undergo hemiscrotectomy after enucleation through a transscrotal access, was considered stage II. All the patients are in first complete remission (mean follow-up, 59 months; range, 8-94). CONCLUSIONS Our experience confirmed the rarity of testicular SCST. They have to be considered in the differential diagnosis of testicular solid masses, taking into account that hormonal signs are present in a minority of cases. All patients were cured with surgery alone. The sparing surgery represented a choice in selected cases.

High-throughput microRNA profiling of pediatric high-grade gliomas

Background High-grade gliomas (HGGs) account for 15% of all pediatric brain tumors and are a leading cause of cancer-related mortality and morbidity. Pediatric HGGs (pHGGs) are histologically indistinguishable from their counterpart in adulthood. However, recent investigations indicate that differences occur at the molecular level, thus suggesting that the molecular path to gliomagenesis in childhood is distinct from that of adults. MicroRNAs (miRNAs) have been identified as key molecules in gene expression regulation, both in development and in cancer. miRNAs have been investigated in adult high-grade gliomas (aHGGs), but scant information is available for pHGGs. Methods We explored the differences in microRNAs between pHGG and aHGG, in both fresh-frozen and paraffin-embedded tissue, by high-throughput miRNA profiling. We also evaluated the biological effects of miR-17-92 cluster silencing on a pHGG cell line. Results Comparison of miRNA expression patterns in formalin versus frozen specimens resulted in high correlation between both types of samples. The analysis of miRNA profiling revealed a specific microRNA pattern in pHGG with an overexpression and a proliferative role of the miR-17-92 cluster. Moreover, we highlighted a possible quenching function of miR-17-92 cluster on its target gene PTEN, together with an activation of tumorigenic signaling such as sonic hedgehog in pHGG. Conclusions Our results suggest that microRNA profiling represents a tool to distinguishing pediatric from adult HGG and that miR-17-92 cluster sustains pHGG.

Primary Rhabdoid Tumour of the Skin in a 14-Month-Old Child

We report on a primary cutaneous rhabdoid tumour in a 14-month-old child, to the best of our knowledge, the second case in the literature. The tumour showed a multipolypoid gross appearance and classical histological features. The neoplastic cells were positive for keratin and vimentin and most were positive for proliferating cell nuclear antigen. The ultrastructural examination revealed typical intracytoplasmic aggregates of intermediate filaments. The tumour showed a very aggressive course, and the child died 5 months after the diagnosis of cerebral metastasis.

Claudin-6 is of limited sensitivity and specificity for the diagnosis of atypical teratoid/rhabdoid tumors.

Recent gene expression microarray analyses have indicated that claudin‐6 is specifically expressed in atypical teratoid rhabdoid tumors (AT/RTs), suggesting a role as a positive diagnostic marker in addition to SMARCB1 (INI1) loss, which is encountered in the majority of AT/RTs. In order to investigate the potential of claudin‐6 as a diagnostic marker, expression was investigated in 59 AT/RTs and 60 other primary central nervous system (CNS) tumors, including primitive neuroectodermal tumors, medulloblastomas, choroid plexus tumors, and both pediatric and adult low‐ and high‐grade gliomas using immunohistochemistry. Claudin‐6 was expressed in 17/59 AT/RTs (29%), but also in a variety of other primary CNS tumors, including 60% of medulloblastomas and 21% of malignant gliomas. Even though high staining scores (2+ or 3+) were more often encountered in AT/RTs (Chi‐square 4.177; P = 0.041), the overall frequency of claudin‐6 staining was not significantly higher in AT/RTs as compared with the other tumors (17/59 vs. 16/60; Chi‐square = 0.328; P = 0.567). In a subgroup of 43 AT/RT patients, of which follow‐up data were available, claudin‐6 expression did not show any correlation with survival. In conclusion, claudin‐6 immunohistochemistry is of limited sensitivity and specificity for the diagnosis of AT/RT and does not correlate with clinical behavior.

Multicystic mesothelial proliferation. Immunohistochemical, ultrastructural and DNA analysis of five cases.

We investigated the clinicopathological findings in five cases of multicystic mesothelial proliferation (MMP). All masses consisted of multiloculated cysts attached to pelvic organs and sometimes growing into the upper abdominal cavity. The cystic spaces were lined by flattened or cuboidal cells. The stroma showed fibrosis, oedema and chronic inflammation. Immunohistochemistry revealed strong positive staining for cytokeratin and epithelial membrane antigen, and focal positivity for vimentin and carcinoembryonic antigen. The endothelial markers were negative. Electron microscopy showed abundant surface microvilli and well-developed basal lamina. DNA analysis identified euploid cell populations in all cases. All but one case had a previous history of abdominal surgery. Despite the worrying appearance the clinical outcome was favourable in all cases; there was one recurrence. Clinical and pathological data support the hypothesis that MMP represent a reactive mesothelial proliferation and not a neoplastic process.

GATA-4 and FOG-2 Expression in Pediatric Ovarian Sex Cord-Stromal Tumors Replicates Embryonal Gonadal Phenotype: Results from the TREP Project

Aim GATA proteins are a family of zinc finger transcription factors regulating gene expression, differentiation and proliferation in various tissues. The expression of GATA-4 and FOG-2, one of its modulators, was studied in pediatric Sex Cord-Stromal tumors of the ovary, in order to evaluate their potential role as diagnostic markers and prognostic factors. Materials and Methods Clinical and histological data of 15 patients, enrolled into the TREP Project since 2000 were evaluated. When available, immunostaines for FOG-2, GATA-4, α-Inhibin, Vimentin and Pancytokeratin were also analyzed. Results In our series there were 6 Juvenile Granulosa Cell Tumors (JGCT), 6 Sertoli-Leydig Cell Tumors (SLCT), 1 Cellular Fibroma, 1 Theca Cell Tumor and 1 Stromal Sclerosing Tumor (SST). Thirteen patients obtained a complete remission (CR), 1 reached a second CR after the removal of a metachronous tumor and 1 died of disease. Inhibin was detectable in 11/15, Vimentin in 13/15, Pancytokeratin in 6/15, GATA-4 in 5/13 and FOG-2 in 11/15. FOG-2 was highly expressed in 5/6 JGCT, while GATA-4 was weakly detectable only in 1 of the cases. SLCT expressed diffusely FOG-2 (4/6) and GATA-4 (3/5). GATA-4 and FOG-2 were detected in fibroma and thecoma but not in the SST. Conclusions Pediatric granulosa tumors appear to express a FOG-2/GATA-4 phenotype in keeping with primordial ovarian follicles. High expression of GATA-4 does not correlate with aggressive behaviour as seen in adults, but it is probably involved in cell proliferation its absence can be associated with the better outcome of JGCT. SLCTs replicate the phenotype of Sertoli cells during embryogenesis in normal testis. In this group, the lack of expression of FOG-2 in tumors in advanced stages might reveal a hypothetical role in inhibiting GATA-4 cell proliferation pathway. In fibroma/thecoma group GATA-4 and FOG-2 point out the abnormal activation of GATA pathway and might be involved in the onset of these tumors.