Tumor-infiltrating T cells and PD-L1 expression in childhood malignant extracranial germ-cell tumors.

Although pediatric malignant extracranial germ-cell tumors (meGCTs) are among the most chemosensitive solid tumors, a group of patients relapse and die of disease. To identify new markers predicting clinical outcome, we examined the prognostic relevance of tumor-infiltrating T lymphocytes (TILs) and the expression of PD-1 and PD-L1 in a cohort of pediatric meGCTs by in situ immunohistochemistry. MeGCTs were variously infiltrated by T cell-subtypes according to the tumor subtype, tumor location and age at diagnosis. We distinguished three different phenotypes: i) tumors not infiltrated by T cells (immature teratomas and half of the yolk sac tumors), ii) tumors highly infiltrated by CD8+ T cells expressing PD-1, which identifies activated tumor-reactive T cells (seminomas and dysgerminomas), iii) tumors highly infiltrated by CD8+ T cells within an immunosuppressive tumor microenvironment characterized by CD4+FOXP3+ Treg cells and PD-L1-expressing tumor cells (embryonal carcinomas, choriocarcinomas and the remaining yolk sac tumors). Tumor subtypes belonging mixed meGCTs were variously infiltrated, suggesting the coexistence of multiple immune microenvironments either facilitating or precluding the entry of T cells. These findings support the hypothesis that TILs influence the development of meGCTs and might be of clinical relevance to improve risk stratification and the treatment of pediatric patients.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

Evaluation of Endoglin (CD105) expression in pediatric rhabdomyosarcoma.

BACKGROUND: The Intratumoral Microvessel Density (IMVD) is commonly used to quantify tumoral vascularization and is usually assessed by pan-endothelial markers, such as CD31. Endoglin (CD105) is a protein predominantly expressed in proliferating endothelium and the IMVD determined by this marker measures specifically the neovascularization. In this study, we investigated the CD105 expression in pediatric rhabdomyosarcoma and assessed the neovascularization by using the angiogenic ratio IMVD-CD105 to IMVD-CD31. METHODS: Paraffin-embedded archival tumor specimens were selected from 65 pediatric patients affected by rhabdomyosarcoma. The expression levels of CD105, CD31 and Vascular Endothelial Growth Factor (VEGF) were investigated in 30 cases (18 embryonal and 12 alveolar) available for this study. The IMVD-CD105 to IMVD-CD31 expression ratio was correlated with clinical and pathologic features of these patients. RESULTS: We found a specific expression of endoglin (CD105) in endothelial cells of all the rhabdomyosarcoma specimens analyzed. We observed a significant positive correlation between the IMVD individually measured by CD105 and CD31. The CD105/CD31 expression ratio was significantly higher in patients with lower survival and embryonal histology. Indeed, patients with a CD105/CD31 expression ratio < 1.3 had a significantly increased OS (88%, 95%CI, 60%-97%) compared to patients with higher values (40%, 95%CI, 12%-67%). We did not find any statistical correlation among VEGF and EFS, OS and CD105/CD31 expression ratio. CONCLUSION: CD105 is expressed on endothelial cells of rhabdomyosarcoma and represent a useful tool to quantify neovascularization in this tumor. If confirmed by further studies, these results will indicate that CD105 is a potential target for combined therapies in rhabdomyosarcoma.

Pediatric adrenocortical neoplasms: immunohistochemical expression of p57 identifies loss of heterozygosity and abnormal imprinting of the 11p15.5.

BACKGROUND: Diagnosis of adrenocortical neoplasms (ACN), in pediatric age, is based on Wieneke criteria. The p57, a cyclin-dependent kinase inhibitor, acts to negatively regulate cell proliferation and is frequently found dysregulated in cancer. The identification of loss of heterozygosity (LOH) of 11p15, containing the p57 gene, could be a tool for differential diagnosis of benign and malignant ACN. METHODS: Immunohistochemistry with anti-p57 and microsatellite markers analysis of 11p15 region to value LOH were made in both ACN and surrounded normal adrenal cortex. RESULTS: Nine ACN, two clinically benign, two uncertain, and five malignant, were diagnosed. Positive p57 cells were evident in normal adrenal cortex and in one histologically benign ACN. A low/absent expression of p57 was documented in eight ACN independently from the classification on the basis of pathological and clinical criteria. Microsatellite marker analysis confirmed the LOH of 11p15 region in the same ACN. CONCLUSION: LOH of 11p15 has a high prognostic value suggesting the p57 gene is important in ACN pathogenesis. Immunohistochemistry for p57 is a simple and cheap tool that can be used to quickly identify LOH of 11p15 in ACN.

IDO1 involvement in mTOR pathway: a molecular mechanism of resistance to mTOR targeting in medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Despite therapeutic advancements, high-risk groups still present significant mortality. A deeper knowledge of the signaling pathways contributing to MB formation and aggressiveness would help develop new successful therapies. The target of rapamycin, mTOR signaling, is known to be involved in MB and is already targetable in the clinical setting. Furthermore, mTOR is a master metabolic regulator able to control cell growth versus autophagy decisions in conditions of amino-acid deprivation that can be due to IDO1 enzymatic activity. IDO1 has been also implicated in the regulation of inflammation, as well as of T cell-mediated immune responses, in a variety of pathological conditions, including brain tumors. In particular, IDO1 induces expansion of regulatory T-cells (Treg), preventing immune response against tumor cells. Analysis of 27 MB tissue specimens for the expression of both mTOR and IDO1 showed their widespread expression in all samples. Testing their cooperation in vitro, a significant involvement of IDO1 in mTOR immunogenic pathway was found, able to counteract the aim of rapamycin treatment. In MB cell lines, inhibition of mTOR strongly induced IDO1 expression and activity, corroborating its ability to recruit Treg cells in the tumor microenvironment. The mTOR/IDO1 cross talk was found to be strictly specific of MB cells. We demonstrated that mTOR pathway cross talks with IDO1 pathway to promote MB immune escape, possibly contributing to failure of mTOR- targeted therapy.